Luminal-like Cells Research Articles

Abstract 3665: Stratifying prostate cancer patients through circulating genes related to prostate cell subtypes, drug targets, and therapeutic resistance

Abstract Stratification remains an obstacle for the optimal treatment of prostate cancer (PCa) patients throughout the treatment trajectory. Targeting the androgen receptor (AR) kills the majority of luminal-like cells, which are AR-positive and express Prostate Specific Antigen (PSA). However, a subset of surviving AR-positive cells develop resistance to treatments while AR-negative cells of the neuroendocrine (NE) or stem-like phenotypes emerge through selection. We reported on the clinical relevance of cell-subtype genes in whole blood RNA of advanced PCa patients. Here, we studied an expanded panel of genes to include drug targets and therapeutic resistance as predictive biomarkers to stratify patients at diagnosis and at the advanced stage of disease.Genes were chosen based on literature review, results of clinical trials in PCa, and PCa transcriptomic data. Technical validation of TaqMan RT-qPCR assays was carried out for each gene, including rigorous testing of reproducibility. Whole blood RNA was extracted from 26 healthy controls with no prostatic disease, 16 patients prior to prostatectomy, and 43 blood samples from 28 metastatic cases. Gene overexpression was defined as the 99.5% confidence interval of expression in controls. Clinical data were retrieved from patients’ charts.A panel of 64 genes was built, showing overexpression in advanced PCa but low or no expression in normal blood (including whole blood, white and red blood cell populations and platelets). Testing in control blood showed no correlation with age. The proportions of patients’ white blood cells did not correlate with gene expression in their blood. Overall, up to 44/64 genes were overexpressed in at least one patient sample. Patients with prostatic intraductal carcinoma at prostatectomy showed more circulating genes, including more NE and stemness genes, and more PCa cell subtypes represented. Intermediate and high-risk patients showed more circulating NE genes. In metastatic patients, signatures of luminal, NE, stemness, and resistance to AR inhibitors or taxanes were associated with progression. PCa-specific luminal genes were associated with shorter overall survival. Treatment resistance genes correlated with lines of treatment and current taxanes. Two targetable NE genes were overexpressed in distinct categories of patients, suggesting that they may benefit from more specific treatments.In conclusion, phenotypic and functional differences in circulating gene patterns of PCa patients correlate with pathological features, treatments, progression. They may be clinically meaningful to stratify patients and predict therapeutic response. Circulating genes encoding drug targets may justify clinical trials to offer personalized treatments and impact on lethal PCa. Citation Format: Seta Derderian, Edouard Jarry, Arynne Santos, Mohanachary Amaravadi, Quentin Vesval, Lucie Hamel, Raphael Sanchez-Salas, Alexis Rompré-Brodeur, Wassim Kassouf, Raghu Rajan, Marie Duclos, Fadi Brimo, Armen Aprikian, Simone Chevalier. Stratifying prostate cancer patients through circulating genes related to prostate cell subtypes, drug targets, and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3665.

Abstract B012: Wnt/Ror2 signaling regulates myoepithelial cell fate and luminal cell proliferation during mammary development

Abstract Background: The mammary gland exhibits extensive proliferative and differentiation potential throughout development. Wnt signaling has been recognized as a crucial player in multiple stages of mammary gland development, including mammary placode specification, gland branching morphogenesis, stem cell maintenance, and alveolar development. In our laboratory, prior research has revealed the importance of the β-catenin-independent noncanonical Wnt receptor, Ror2, in branching morphogenesis, cell differentiation, and actin-cytoskeletal dynamics within the mammary epithelium. However, the specific roles of Ror2 in the myoepithelial and luminal cell compartments have yet to be fully elucidated. Methods and Results: In this study, we investigated the functional role of Ror2 in these two distinct cell populations by developing mouse models with lineage-specific deletion of Ror2 in myoepithelial versus luminal epithelial compartments. To achieve this, we generated two tamoxifen-inducible, p63- and Keratin 8-specific Ror2 knockout mouse models, which carried a ROSA26-mT/mG fluorescent reporter for mammary myoepithelial cells and luminal cells, respectively. We induced postnatal Ror2 deletion at week 4 or 6 and assessed the mammary glands 2 to 4 weeks after Ror2 deletion. Through whole-mount imaging of mammary glands, we observed a significant increase in branching morphogenesis when Ror2 was deleted in the myoepithelial compartment, which was further confirmed in 3D branching assays in vitro. RNA-sequencing analysis revealed novel transcriptional alterations encompassing altered morphogenesis, cell differentiation, and intermediate filament organization upon Ror2 loss. Additional gene expression validation and immunofluorescence demonstrated altered myoepithelial cell fate specification, leading to a shift to a luminal-like cell fate, along with altered cell-cell adhesion among epithelial populations and abnormal branching morphogenesis within the developing gland. Moreover, luminal-specific deletion of Ror2 resulted in increased proliferation and abnormal lumen formation, both in vivo and in vitro. Conclusion: Our findings from these tissue-specific knockout mouse models highlight the critical role of the noncanonical Wnt receptor, Ror2, in regulating mammary epithelial cell fate, branching morphogenesis, and proliferation. These results also provide intriguing insights into the coordination between canonical and noncanonical Wnt signaling in mammary gland development, with potential implications for understanding breast cancer tumorigenesis and heterogeneity (Supported by NIH CA016303-46 and CPRIT RP210027). Citation Format: Hongjiang Si, Jeffrey Rosen, Chad Creighton, Kevin Roarty. Wnt/Ror2 signaling regulates myoepithelial cell fate and luminal cell proliferation during mammary development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B012.

Metabolic switch regulates lineage plasticity and induces synthetic lethality in triple-negative breast cancer

Metabolic reprogramming is key for cancer development, yet the mechanism that sustains triple-negative breast cancer (TNBC) cell growth despite deficient pyruvate kinase M2 (PKM2) and tumor glycolysis remains to be determined. Here, we find that deficiency in tumor glycolysis activates a metabolic switch from glycolysis to fatty acid β-oxidation (FAO) to fuel TNBC growth. We show that, in TNBC cells, PKM2 directly interacts with histone methyltransferase EZH2 to coordinately mediate epigenetic silencing of a carnitine transporter, SLC16A9. Inhibition of PKM2 leads to impaired EZH2 recruitment to SLC16A9, and in turn de-represses SLC16A9 expression to increase intracellular carnitine influx, programming TNBC cells to an FAO-dependent and luminal-like cell state. Together, these findings reveal a new metabolic switch that drives TNBC from a metabolically heterogeneous-lineage plastic cell state to an FAO-dependent-lineage committed cell state, where dual targeting of EZH2 and FAO induces potent synthetic lethality in TNBC.

Expression of type VI collagen α3 chain in canine mammary carcinomas

This study aimed to investigate the expression of type VI collagen α3 chain (COL6a3) in neoplastic cells of canine mammary gland carcinomas (CMGCs) using immunohistochemistry (IHC) and to evaluate the association between COL6a3 expression and tumor histological features, histological grades, and the differentiation status of neoplastic epithelial cells. COL6a3 expression in carcinoma cells was significantly associated with histologically low malignancy and low mitotic indices. In addition, COL6a3+ carcinoma cells were more frequently detected in simple carcinomas (tubular and tubulopapillary types) than in solid carcinomas. These findings indicate that reduced expression of COL6a3 in carcinoma cells contributes to the malignant phenotype in CMGCs. We also showed that COL6a3 expression in the carcinoma cells was more frequently detected in CK19+/CD49f + and/or CK19+/CK5+ tumors. In addition, COL6a3+/CK19+/CD49f + and COL6a3+/CK19+/CK5+ tumors consisted of CK19+/CD49f + and CK19+/CD49f- cells, and CK19+/CK5+ and CK19+/CK5- cells, respectively. Most of these tumors more frequently expressed GATA3, but not Notch1. These results indicate that COL6a3 is expressed in CMGCs containing both luminal progenitor-like and mature luminal-like cells and showing differentiation ability into mature luminal cells. It is possible that COL6 may be involved in the differentiation of luminal progenitor-like carcinoma cells into mature luminal-like carcinoma cells in CMGCs, which may suppresses the development of malignant phenotypes in CMGCs.

MP06-08 SINGLE-CELL SEQUENCING OF PARENTAL BLADDER CANCER AND SUBSEQUENT ORGANOIDS REVEALS DIFFERENTIAL CELL PRESERVATION

You have accessJournal of UrologyCME1 May 2022MP06-08 SINGLE-CELL SEQUENCING OF PARENTAL BLADDER CANCER AND SUBSEQUENT ORGANOIDS REVEALS DIFFERENTIAL CELL PRESERVATION Roland Seiler, Martina Minoli, Franziaks Singer, Anne Bertolini, Ulrike Menzel, Christian Beisel, and Marianna Kruithof-de Julio Roland SeilerRoland Seiler More articles by this author , Martina MinoliMartina Minoli More articles by this author , Franziaks SingerFranziaks Singer More articles by this author , Anne BertoliniAnne Bertolini More articles by this author , Ulrike MenzelUlrike Menzel More articles by this author , Christian BeiselChristian Beisel More articles by this author , and Marianna Kruithof-de JulioMarianna Kruithof-de Julio More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Molecular heterogeneity of bladder cancer (BC) is affecting response to treatment. To test treatment response, several patient derived models have been established that may vary in their suitability to test targets. We have previously established patient derived organoids (PDO) from BC patients and found a highly conserved genomic landscape. In this analysis, we aim to investigate the cellular composition and pathway activities of this patient derived model. METHODS: We generated PDOs from more than 35 BC specimens. Based on the marker expression, we selected 5 pairs of BC and subsequent PDO to include different molecular subtypes (e.g. luminal-, basal-like). Single-cell suspensions were directed to scRNA-seq technology from 10x Genomics. For the analysis, unsupervised clustering, gene expression, as well as pathway analysis were used to compare the cell type composition and pathway activity. RESULTS: Overall, we observed a high degree of heterogeneity of cell type composition between BCs, between PDOs, as well as between BCs to subsequent PDOs. Unsupervised clustering separated cancerous- as well as non-cancerous cells in BCs and PDOs in individual clusters. In most BC, both luminal-like and basal-like cells were detected. In the subsequent PDOs however, luminal-like cells were present with a higher proportion compared to basal-like cells. This was true for different luminal-like subcategories (lum-pap and lum-nonspecified). While immune cells (e.g. B-cells, CD4+ and CD8+ T-cells) were present in both BC and PDO samples, stromal cells (e.g. smooth muscle and endothelial cells) were absent in PDOs. Consequently, immune- and cancer cell related pathways such as interferon_gamma_response or FGFR3_signature were active in both BC and PDO. Contrarily, pathways related to stroma interactions such as angiogenesis or epithelial_mesenchymal_transition were less active in PDOs. CONCLUSIONS: Single-cell transcriptomic profiling of parental BCs and subsequent PDOs revealed a high degree of heterogeneity in both the tumor and organoid cellular composition. Our data show preservation of immune cells in PDOs and thus suggest that organoids may even allow to investigate cellular interactions between cancer and immune cells. On the other hand, since stromal cells were mainly lost in PDOs, other models may be needed to investigate angiogenesis or epithelial mesenchymal transition. Source of Funding: SNSF Grant Nr. 310030 Novartis Grant #19C200grant #2017-510 of the Strategic Focal Area “Personalized Health and Related Technologies (PHRT)” of the ETH Domain © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e79 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Roland Seiler More articles by this author Martina Minoli More articles by this author Franziaks Singer More articles by this author Anne Bertolini More articles by this author Ulrike Menzel More articles by this author Christian Beisel More articles by this author Marianna Kruithof-de Julio More articles by this author Expand All Advertisement PDF DownloadLoading ...

Prostate luminal progenitor cells: from mouse to human, from health to disease.

Stem and progenitor cells of the adult prostate epithelium have historically been believed to reside mainly or exclusively within the basal cell compartment and to possess basal-like phenotypic characteristics. Within the past decade, evidence of the existence of luminal epithelial cells exhibiting stem/progenitor properties has been obtained by lineage tracing and by functional characterization of sorted luminal-like cells. In 2020, the boom of single-cell transcriptomics led to increasingly exhaustive profiling of putative mouse luminal progenitor cells and, importantly, to the identification of cognate cells in the human prostate. The enrichment of luminal progenitor cells in genetically modified mouse models of prostate inflammation, benign prostate hypertrophy and prostate cancer, and the intrinsic castration tolerance of these cells, suggest their potential role in prostate pathogenesis and in resistance to androgen deprivation therapy. This Review bridges different approaches that have been used in the field to characterize luminal progenitor cells, including the unification of multiple identifiers employed to define these cells (names and markers). It also provides an overview of the intrinsic functional properties of luminal progenitor cells, and addresses their relevance in mouse and human prostate pathophysiology.

Tracing tumorigenesis in a solid tumor model at single-cell resolution

Characterizing the complex composition of solid tumors is fundamental for understanding tumor initiation, progression and metastasis. While patient-derived samples provide valuable insight, they are heterogeneous on multiple molecular levels, and often originate from advanced tumor stages. Here, we use single-cell transcriptome and epitope profiling together with pathway and lineage analyses to study tumorigenesis from a developmental perspective in a mouse model of salivary gland squamous cell carcinoma. We provide a comprehensive cell atlas and characterize tumor-specific cells. We find that these cells are connected along a reproducible developmental trajectory: initiated in basal cells exhibiting an epithelial-to-mesenchymal transition signature, tumorigenesis proceeds through Wnt-differential cancer stem cell-like subpopulations before differentiating into luminal-like cells. Our work provides unbiased insights into tumor-specific cellular identities in a whole tissue environment, and emphasizes the power of using defined genetic model systems.

Abstract B031: CREB1 and ATF1 differentially regulate terminal prostate luminal differentiation by controlling the timing of ING4 expression, while CREB1 prevents ING4 expression upon PTEN loss in prostate cancer

Abstract Many genes aberrantly expressed in prostate cancer are involved in normal basal to luminal cell differentiation. We previously demonstrated that transient ING4 expression is required for luminal cell differentiation and is downregulated in ~60% of primary prostate tumors. We further demonstrated in a primary prostate cancer model overexpressing ERG, MYC, and shPTEN (EMP) that loss of PTEN was responsible for ING4 loss. Furthermore, half of the human tumor samples that lose ING4 have also lost PTEN. However, we did not know how PTEN loss inhibits ING4 expression. Utilizing our in vitro differentiation model, whereby prostate basal epithelial cells (iPrEC) treated with KGF and androgen induce a suprabasal layer of luminal-like cells, and RNA-seq we identified transcriptional nodes required for luminal differentiation. Differentially expressed genes were analyzed by GeneGo to identify enriched transcription-factor signatures. Of the ~600 differentially regulated genes during differentiation, the largest signature (29% of genes) was CREB/ATF targets. Induction of Blimp1, Claudin1, and Plk2 and inhibition of Chek1 were further validated by qRT-PCR and immunoblotting. CREB/ATF bind constitutively to open chromatin CRE elements in the promoters of genes and are activated through signaling-induced phosphorylation at Ser133 by kinases, including AKT. We found that both CREB1 and ATF1 are inducibly phosphorylated midway through luminal differentiation, with ATF1 preceding CREB1. Knockdown of CREB1 with shRNA increased ING4, accelerated differentiation, and induced premature luminal cell death. Conversely, knockdown of ATF1 blocked ING4 induction and prevented suprabasal layer formation. CREB1/ATF1 ChIP was enriched at the ING4 promoter at mid-differentiation, when ING4 expression peaks. Additionally, CREB1/ATF1 was constitutively bound to the promoter of JFK, an E3-ligase that targets ING4 and whose mRNA levels increase during differentiation. Thus, we propose that ATF1 is required to induce ING4 transcription, while CREB1 suppresses ING4 and simultaneously activates its E3-ligase to tightly control the timing of ING4 expression. We compared the gene signature of luminal cells to that of the tumorigenic EMP cells and, surprisingly, found that 30% of the differentially expressed genes were also CREB/ATF targets. However, there is less than 10% overlap in these targets, indicating that CREB/ATF control distinct subsets of genes in differentiated luminal cells versus cancer cells. Some of EMP-specific CREB/ATF targets included GATA2, TWIST1, Necdin, and PPM1F, which were further validated by qRT-PCR and immunoblotting. CREB1 and ATF1 were highly phosphorylated in EMP cells and knockdown of CREB1 restored ING4 expression and suprabasal formation. Our working model is that AKT activation upon PTEN loss in transiently differentiating luminal cells results in premature and constitutive activation of CREB1/ATF1 bound to genes prior to induction of the ING4 chromatin switch. This prevents ING4 induction and the chromatin rearrangements required for terminal differentiation. In normal PrECs, CREB/ATF1 activation is tightly controlled by as yet undetermined factors and is only permitted when the proper CRE binding sites are exposed. This model helps to explain how loss of PTEN disrupts luminal cell terminal differentiation to promote prostate cancer oncogenesis. Citation Format: McLane Watson, Penny Berger, Sander Frank, Mary Winn, Cindy Miranti. CREB1 and ATF1 differentially regulate terminal prostate luminal differentiation by controlling the timing of ING4 expression, while CREB1 prevents ING4 expression upon PTEN loss in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B031.

Abstract 1729: Detection of gene expression biomarkers from enriched CTC preparations

Abstract Circulating tumor cells (CTCs) offer an opportunity for non-invasive characterization of metastatic cancer, including the ability to follow subtype evolution, development of resistance, tissue homing biomarkers, and changes in cell signaling mechanisms required for metastasis and continued growth and survival of the tumor. Analysis of CTCs and translation of a CTC assay into a clinical test has been difficult due to issues of obtaining high purity, high yield preparations and/or isolating individual CTCs. This is further compounded by the heterogeneity of CTCs, hurdles in profiling their molecular signatures, and difficulty correlating CTC number/subtype to actionable therapy. We pursued a detailed analysis of CTC gene expression using whole transcriptome TempO-Seq targeted sequencing, a highly sensitive, direct lysis, addition only assay. As this assay does not require purified CTC isolation or RNA extraction, there is no risk of CTC nor RNA loss. We exploited the single cell sensitivity of TempO-Seq to profile enriched CTC preparations prepared by RosetteSep gradient centrifugation, allowing us to characterize the phenotypes of CTCs within a high background of normal blood cells. To handle this large and potentially overwhelming background we developed and exploited a proprietary method of sample normalization that increased the sensitivity and consistency of CTC gene signature measurements. We demonstrated that the TempO-Seq assay could detect a single MCF7 (EpCAM+) Luminal-like cell in a background of 1,000 MDA MB 231 (EpCAM-) Basal-like cells (positive for an Endothelial Mesenchymal Transition, EMT), and vice versa. Next, we prepared mixtures of different proportions of these cultured cells spiked into normal blood, enriched the model “CTCs” using RosetteSep, and profiled the bulk preparation. Proportional discrimination of cell-specific signatures were obtained with as few as 40 spike-in cells. Finally, we profiled enriched CTC preparations from the blood of patients with metastatic breast cancer. The results demonstrated that the expression of HER2, ER, and EpCAM could be measured as well as biomarkers of drug resistance, bone/brain homing, EMT, and all 50 Hallmark cell signaling pathways. TempO-Seq EpCAM gene level correlated with the percent EpCAM+ cells measured by flow analysis while EMT signature levels correlated inversely. Patients with HER2- primary tumor and HER2+ CTCs could be identified, as could patients with elevated chemotherapy resistance pathways. These data demonstrate that it is possible to profile CTC phenotypes that are important in metastases and useful for selecting therapy using a bulk CTC preparation combined with the sensitivity of the TempO-Seq whole transcriptome assay. This suggests the opportunity for the development of a predictive test using currently available purification platforms without need for expensive specialized equipment or separation into single cells. Citation Format: Bruce Seligmann, Marilyn Marron, Milos Babic, Elliot Imler, Peter Shepard, Pavani Chalasani, Alison Stopeck, Joanne Yeakley. Detection of gene expression biomarkers from enriched CTC preparations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1729. doi:10.1158/1538-7445.AM2017-1729

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.

Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity.

Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes. PIK3CA and P53 (also known as TP53) are the two most frequently mutated genes and are associated with different types of human breast cancers. The cellular origin and the mechanisms leading to PIK3CA-induced tumour heterogeneity remain unknown. Here we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and the impact of mutations in this gene on tumour heterogeneity. Surprisingly, oncogenic Pik3ca(H1047R) mutant expression at physiological levels in basal cells using keratin (K)5-CreER(T2) mice induced the formation of luminal oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive tumours, while its expression in luminal cells using K8-CReER(T2) mice gave rise to luminal ER(+)PR(+) tumours or basal-like ER(-)PR(-) tumours. Concomitant deletion of p53 and expression of Pik3ca(H1047R) accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3ca(H1047R) in unipotent basal cells gave rise to luminal-like cells, while its expression in unipotent luminal cells gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that underwent cell fate transition upon Pik3ca(H1047R) expression in unipotent progenitors demonstrated a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures characteristic of the different cell fate switches that occur upon Pik3ca(H1047R) expression in basal and luminal cells, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3ca(H1047R) activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation.

Abstract 4207: Secreted proteins from breast cancer cell lines as a source of cancer biomarkers

Abstract Introduction: Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and microscopic markers used for prognostic information and treatment decisions. Traditionally, most biomarkers are based on tissue samples. To increase efficiency, precise liquid biopsies are needed to detect breast cancer at an early stage including its molecular phenotype, to monitor disease progression, and to predict treatment response. Cell line secretomes are enriched with proteins already linked to tumorigenesis, many of which will be present in biological fluids. Methods: Here, we studied the secreted proteins in conditioned media (CM) from two basal-like (HS-578, MB-231) and two luminal-like breast cancer cell lines (MCF-7, BT-474), using liquid chromatography-tandem mass spectrometry. The resulting data were analyzed using SearchGUI, PeptideShaker, Progenesis software and DAVID. Results: In total, 954 proteins were identified, of which 93 significantly differentially abundant (P-value ≤ 0.05 Mann-Whitney U, two or more peptides quantified) in the CM between basal-like and luminal-like cell lines. 76 proteins were more abundant in the CM from basal-like cell lines, including several proteins related to angiogenesis and extra-cellular matrix remodeling. Conclusion: This study shows the potential for using the secretome of breast cancer cell lines combined with mass-spectrometry for biomarker discovery. Citation Format: Even Birkeland, Monica Mannelqvist, Lars Andreas Akslen. Secreted proteins from breast cancer cell lines as a source of cancer biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4207. doi:10.1158/1538-7445.AM2015-4207

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.

Prostate cancer originating in basal cells progresses to adenocarcinoma propagated by luminal-like cells

The relationship between the cells that initiate cancer and the cancer stem-like cells that propagate tumors has been poorly defined. In a human prostate tissue transformation model, basal cells expressing the oncogenes Myc and myristoylated AKT can initiate heterogeneous tumors. Tumors contain features of acinar-type adenocarcinoma with elevated eIF4E-driven protein translation and squamous cell carcinoma marked by activated beta-catenin. Lentiviral integration site analysis revealed that alternative histological phenotypes can be clonally derived from a common cell of origin. In advanced disease, adenocarcinoma can be propagated by self-renewing tumor cells with an androgen receptor-low immature luminal phenotype in the absence of basal-like cells. These data indicate that advanced prostate adenocarcinoma initiated in basal cells can be maintained by luminal-like tumor-propagating cells. Determining the cells that maintain human prostate adenocarcinoma and the signaling pathways characterizing these tumor-propagating cells is critical for developing effective therapeutic strategies against this population.

Abstract 5209: Prostate cancer progenitor cells have distinct DNA methylation profile which changes upon differentiation

Abstract Prostate cancer is phenotypically, genetically and epigenetically heterogeneous. In the present study we have dissected the epigenetic heterogeneity of prostate cancer by analyzing DNA methylation patterns in pure subpopulations of primary cancer cells, on the basis of their differentiation status. GSTP1, an enzyme involved in intracellular detoxification, is highly expressed in prostate basal epithelial cells but is down-regulated in luminal cells by a mechanism independent of DNA methylation. The GSTP1 promoter becomes frequently hypermethylated in prostate cancer, where the majority of cells bear a luminal-like phenotype. However, a small subpopulation of basal cells (&amp;lt;1%) persists within prostate cancer and is hypothesized to contain stem-like cells that give rise to aberrantly differentiated cancer cells. By analyzing the DNA methylation patterns in basal and luminal like prostate cancer cells, it has been possible to unveil the origin of promoter hypermethylation in prostate cancer that ultimately leads to epigenetic heterogeneity. In established cell lines, GSTP1 was actively transcribed (measured by RT-PCR) and not hypermethylated (measured by pyrosequencing methylation assay) in basal-like cancer cells, while it was hypermethylated and down-regulated in luminal-like cancer cells. MACs selection of Lin−/CD31−/CD24+ cells from prostate primary tissues highly enriched for AR+/KRT8+/GSTP1low cells bearing a luminal-like phenotype, while generation of prostate primary epithelial cultures gave rise to cells with basal phenotype. In luminal-like cells, GSTP1 was hypermethylated in cancer samples compared to benign controls. However, no significant hypermethylation of GSTP1 was found in the basal-like cells, where the gene was actively expressed in both benign and cancer samples. Lack of GSTP1 promoter methylation was also found in tumor xenografts generated in Rag2−/+gammaC−/+ mice from primary prostate cancer tissues. These xenografts do not undergo complete differentiation and show an intermediate phenotype expressing both basal and luminal markers. Moreover, in BPH-1 cells, a fast cycling immortalized cell line, expression and promoter methylation of GSTP1 correlated with the differentiation status of the cells, being hypermethylated in more differentiated cells. Our results strongly indicate that within prostate cancer there is a subpopulation of undifferentiated basal-like cells that do not hypermethylate the GSTP1 promoter. We hypothesize that these cells can differentiate into luminal-like cancer cells, which down-regulate GSTP1 and hypermethylate its promoter as a consequence of aberrant proliferation. Future work will include determination of the mechanism of GSTP1 down-regulation in normal and malignant prostate epithelial differentiation, and whether this mechanism is shared with other genes frequently hypermethylated in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5209. doi:1538-7445.AM2012-5209

Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity

The majority of human breast cancers exhibit luminal epithelial differentiation. However, most aggressive behavior, including invasion and purported cancer stem cell activity, are considered characteristics of basal-like cells. We asked the following questions: Must luminal-like breast cancer cells become basal-like to initiate tumors or to invade? Could luminally differentiated cells within a basally initiated hierarchy also be tumorigenic? To answer these questions, we used rare and mutually exclusive lineage markers to isolate subsets of luminal-like and basal-like cells from human breast tumors. We enriched for populations with or without prominent basal-like traits from individual tumors or single cell cloning from cell lines and recovered cells with a luminal-like phenotype. Tumor cells with basal-like traits mimicked phenotypic and functional behavior associated with stem cells assessed by gene expression, mammosphere formation and lineage markers. Luminal-like cells without basal-like traits, surprisingly, were fully capable of initiating invasive tumors in NOD SCID gamma (NSG) mice. In fact, these phenotypically pure luminal-like cells generated larger and more invasive tumors than their basal-like counterparts. The tumorigenicity and invasive potential of the luminal-like cancer cells relied strongly on the expression of the gene GCNT1, which encodes a key glycosyltransferase controlling O-glycan branching. These findings demonstrate that basal-like cells, as defined currently, are not a requirement for breast tumor aggressiveness, and that within a single tumor there are multiple "stem-like" cells with tumorigenic potential casting some doubt on the hypothesis of hierarchical or differentiative loss of tumorigenicity.

In Situ Proximity Ligation Detection of c-Jun/AP-1 Dimers Reveals Increased Levels of c-Jun/Fra1 Complexes in Aggressive Breast Cancer Cell Lines in Vitro and in Vivo

Genetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1) exhibit specific and critical functions in the regulation of cell proliferation, differentiation, and survival. For instance, the ratio between c-Jun/c-Fos and c-Jun/ATF2 dimers in the cell can be a determining factor in the cellular response to oncogenic or apoptotic stimuli. Until recently, no methods were available to detect endogenous AP-1 complexes in cells and tissues in situ. Here, we validated the proximity ligation assay (PLA) for its ability to specifically visualize and quantify changes in endogenous c-Jun/c-Fos, c-Jun/ATF2, and c-Jun/Fra1 complexes by using, among others, partner-selective c-Jun mutants. Furthermore, we examined the levels of c-Jun/AP-1 dimers in cell lines representing different types of human breast cancer and found that aggressive basal-like breast cancer cells can be discriminated from much less invasive luminal-like cells by PLA detection of c-Jun/Fra1 rather than of c-Jun/ATF2 and c-Jun/c-Fos. Also in tumor tissue derived from highly metastatic basal-like MDA-MB231 cells, high levels of c-Jun/Fra1 complexes were detected. Together, these results demonstrate that in situ PLA is a powerful diagnostic tool to analyze and quantify the amounts of biologically critical AP-1 dimers in fixed cells and tissue material.

Protein Profiling of Human Breast Tumor Cells Identifies Novel Biomarkers Associated with Molecular Subtypes

Molecular subtypes of breast cancer with relevant biological and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal-like subtypes. To investigate the ability of mass spectrometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we performed a SELDI-TOF MS-based protein profiling of human breast cell lines (BCLs). Triton-soluble proteins from 27 BCLs were incubated with ProteinChip arrays and subjected to SELDI analysis. Unsupervised global hierarchical clustering spontaneously discriminated two groups of BCLs corresponding to "luminal-like" cell lines and to "basal-like" cell lines, respectively. These groups of BCLs were also different in terms of estrogen receptor status as well as expression of epidermal growth factor receptor and other basal markers. Supervised analysis revealed various protein biomarkers with differential expression in basal-like versus luminal-like cell lines. We identified two of them as a carboxyl terminus-truncated form of ubiquitin and S100A9. In a small series of frozen human breast tumors, we confirmed that carboxyl terminus-truncated ubiquitin is observed in primary breast samples, and our results suggest its higher expression in luminal-like tumors. S100A9 up-regulation was found as part of the transcriptionally defined basal-like cluster in DNA microarrays analysis of human tumors. S100A9 association with basal subtypes as well as its poor prognosis value was demonstrated on a series of 547 tumor samples from early breast cancer deposited in a tissue microarray. Our study shows the potential of integrated genomics and proteomics profiling to improve molecular knowledge of complex tumor phenotypes and identify biomarkers with valuable diagnostic or prognostic values.

Establishment, growth and in vivo differentiation of a new clonal human cell line, EM-G3, derived from breast cancer progenitors

A new clonal cell line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The line consisted of cuboidal cells with occasional appearance of more differentiated branched cells apparently involved in cell-to-cell communication. The EM-G3 cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins alpha-6, beta-1, and protein CD44 by cDNA array also pointed to the character of basal/stem cells. In contrast, dominant cells in the human original tumor showed the luminal character (K18+, K19+, K5-, K14-, and p63-). However, cells with the immunocytochemical profile similar to that of cultured EM-G3 cells were found in minor clusters in the patient's tumor sections. The EM-G3 cells formed limited tumors in nu/nu mice. The cells in mouse tumors were organized in primitive ductal-like structures consisting of 1-3 large central luminal-like cells (EMA+) surrounded by peripheral myoepithelial-like cells (p63+/EMA-). The large central cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 cells were derived from a premalignant population of common progenitors of luminal and myoepithelial cells that were immortalized in an early stage of tumorigenesis.