Luminal Population Research Articles

Emergence of rifampin-resistant staphylococci after rifaximin administration in cirrhotic patients.

ObjectivesRifaximin, a poorly absorbed antibiotics, has gut-specific therapeutic effects. Although frequently prescribed to manipulate intestinal luminal bacterial population in various diseases, the possible induction of antibacterial cross-resistance to a target pathogen is a major concern in long-term rifaximin administration. We aimed to evaluate whether rifampin-resistant staphylococci could evolve after rifaximin treatment in cirrhotic patients.MethodA total of 25 cirrhotic patients who were administered rifaximin for the prevention of hepatic encephalopathy were enrolled. Swabs from both hands and the perianal skin were acquired on day 0 (before rifaximin treatment), period 1 (1–7 weeks after treatment), and period 2 (8–16 weeks after treatment) the staphylococcal strain identification and rifampin-resistance testing.ResultsA total of 198 staphylococcal isolates from 15 species were identified. Staphylococcus epidermidis was isolated most frequently, and Staphylococcus haemolyticus was the most common resistant species both from hands and perianal skin. Eleven patients (44.0%) developed rifampin-resistant staphylococcal isolates in period 1. Among these patients, only six (54.5%) were found to have rifampin-resistant isolates in period 2, with no significant infectious events. Rifampin-resistant staphylococcal isolates were more frequently found in perianal skin than from the hands. No patients acquired a newly resistant strain in period 2.ConclusionsAbout one-half of cirrhotic patients in this study developed rifampin-resistant staphylococcal isolates after rifaximin treatment. Although the resistant strains were no longer detected in about half of the patients in the short-term, the long-term influence of this drug treatment should be determined.

The Gastric and Intestinal Microbiome: Role of Proton Pump Inhibitors.

The discovery of Helicobacter pylori and other organisms colonizing the stomach and the intestines has shed some light on the importance of microbiome in maintaining overall health and developing pathological conditions when alterations in biodiversity are present. The gastric acidity plays a crucial role in filtering out bacteria and preventing development of enteric infections. In this article, we discuss the physiology of gastric acid secretion and bacterial contribution to the composition of gastric and intestinal barriers and review the current literature on the role of proton pump inhibitors (PPIs) in the microbial biodiversity of the gastrointestinal tract. Culture-independent techniques, such as 16S rRNA sequencing, have revolutionized our understanding of the microbial biodiversity in the gastrointestinal tract. Luminal and mucosa-associated microbial populations are not identical. Streptococcus is overrepresented in the biopsies of patients with antral gastritis and may also be responsible for the development of peptic ulcer disease. The use of PPIs favors relative streptococcal abundance irrespective of H. pylori status and may explain the persistence of dyspeptic symptoms in patients on PPI therapy. Increased risk of enteric infections has also been seen in patients taking PPIs. The overuse of PPIs leads to significant shift of the gastrointestinal microbiome towards a less healthy state. With the advent of PPIs, many studies have demonstrated the significant changes in the microbial composition of both gastric and intestinal microbiota. Although they are considered relatively safe over-the-counter medications, PPIs in many cases are over- and even inappropriately used. Future studies assessing the safety of PPIs and their role in the development of microbiome changes should be encouraged.

Abstract 1908: FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

Abstract The fibroblast growth factor receptor (FGFR) family (FGFR1-4) of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. In particular, FGFR1 and FGFR2, which are found in the terminal end buds of developing mammary ducts, play a role in mammary development and glandular morphogenesis involving the regulation of mammary stem cells (MaSCs) in mice. As such, a number of FGFR inhibitors are being tested in preclinical studies and clinical trials for anti-tumor properties. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we aimed to investigate the anti-cancer benefits of AZD4547, a small molecule inhibitor of FGFR1-3, on ErbB2-overexpressing breast cancer models. We particularly focus on the effects of AZD4547 on MaSCs and tumor-initiating cells (TICs) in the premalignant tissues of MMTV-ErbB2 transgenic mice. We first demonstrated the anti-proliferative effects of AZD4547 (1-5 µM) on human ErbB2-overexpressing breast cancer cell lines. We further showed that AZD4547 confers potent inhibition of the stemness of these breast cancer cells, as indicated by significant depletion of ALDH+ cells and impaired tumorsphere formation. To study the in vivo effects of AZD4547 on the stemness of mammary epithelial cells (MECs), MMTV-ErbB2 transgenic mice were administered AZD4547 (2-6 mg/kg/day) for 10 weeks (weeks 8-18 of age) during the ‘risk window’ for mammary tumor development. Histopathological analysis indicated that AZD4547 significantly inhibited ductal branching and MEC proliferation. To examine the effect of AZD4547 on MEC subpopulations and tissue hierarchy dynamics in the premalignant mammary tissues of this model, we performed flow cytometry analyses on the primary MECs using CD24/CD49f and CD61/CD49f cell surface markers. The results showed that AZD4547 treatment substantially reduced MaSC-derived luminal and myoepithelial cell populations. AZD4547 also selectively suppressed the CD61highCD49fhigh cell population, which is enriched with luminal progenitor cells that give rise to TICs during MMTV-ErbB2 mammary tumor development. Mammosphere and colony-forming cell (CFC) assays on primary MECs demonstrated that the stemness of these cells was also blocked by AZD4547 prior to malignant transformation. Consistently, AZD4547 inhibited the anchorage-independent growth of cells from spontaneous tumors. Moreover, we demonstrated that AZD4547 downregulated multiple pathways, including the inactivation of FGFR, EGFR, and Wnt/β-catenin signaling. Collectively, the morphogenic, MaSC/TIC, and signaling regulation associated with AZD4547 treatment provides critical evidence for AZD4547 as a breast cancer preventative and therapeutic agent, which ultimately reveals clues for more effective eradication of refractory mammary tumors. Citation Format: Qingxia Zhao, Amanda B. Parris, Erin W. Howard, Ming Zhao, Ying Xing, Zhikun Ma, Xiaohe Yang. FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice [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 1908. doi:10.1158/1538-7445.AM2017-1908

Abstract 4775: PTHrP regulate CSC and promote EMT in the PyMT model of breast cancer progression

Abstract PTH-related peptide (PTHrP) was first discovered in cancer patients as the primary cause of humoral hypercalcemia of malignancy (HHM). We previously showed that PTHrP ablation in the MMTV-PyMT murine model of breast cancer progression can dramatically prolongs tumor latency, slows tumor growth & metastases suggesting that PTHrP may regulate cancer stem cells (CSCs). CSCs & epithelial-mesenchymal transition (EMT) have been recognized as driving forces in tumor initiation & progression, & have been characterized previously using ALDH & CD49/EpCAM markers. Here, we aim to determine the role of PTHrP in CSCs & EMT regulation. For this purpose, we analyzed tumor cells derived from Pthrpflox/flox;Cre- (Control) & Pthrpflox/flox;Cre+ (KO) tumors. Cells were incubated with ALDH reagent followed by CD49f/EpCAM antibody labelling & subjected to FACS analysis. Using CD49f/EpCAM labelling, cells separated into two distinct populations, luminal progenitor cells (CD49f+EpCAM+) & EMT cells (CD49f+EpCAM-). In control tumors, ~85% of cells were characterized as luminal progenitor and ~15% as EMT. In contrast, in KO tumors, ~97% of cells were characterized as luminal progenitor whereas only ~2% were EMT. Using ALDH staining alone, ~8% & ~10% of the cells from the control & KO tumor were ALDH+, respectively. Furthermore, the majority of the ALDH+ cells were predominately contained within the luminal progenitor population in both the control & KO tumors cells. Next, we confirmed the dividing capacity of the two cell populations using a lipophilic fluorescent dye, PKH26. As expected, luminal progenitor cells exhibited a fast-dividing phenotype, whereas EMT cells displayed a slow dividing phenotype. However, using mammosphere assay, we did not see any significant difference in mammosphere forming efficiency between control & KO tumor. In summary, PTHrP ablation significantly diminishes the EMT population in KO compared to the control tumor. In conclusion, PTHrP may shift the tumor cell phenotype towards EMT, which suggests a mechanism underlying PTHrP - driven metastases. Citation Format: Rui Zhang, Jiarong Li, Andrew C. Karaplis, Richard Kremer. PTHrP regulate CSC and promote EMT in the PyMT model of breast cancer progression [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 4775. doi:10.1158/1538-7445.AM2017-4775

Evolution of overall survival according to year of diagnosis (2008-2014) and subtypes, among 16703 metastatic breast cancer (MBC) patients included in the real-life "ESME" cohort.

1078 Background: Real-life data may help checking that public investments match closely medical needs. During the last decade, several drugs have been released on the market for MBC on the basis of a potential impact on overall survival (OS). Based on the large real-life ESME cohort, we aimed to describe the time evolution of MBC OS according to main phenotypes. Methods: ESME is a unique MBC national cohort including all consecutive patients (pts) who initiated treatment for MBC between 1/01/08 and 31/12/14 in the 18 French comprehensive cancer centres. ESME collects retrospective data using clinical trial-like methodology including quality assessments. Database lock was 8/12/2016. Primary objective was the impact of year of MBC diagnosis on OS. Multivariate Cox regressions were used with adjustment for main prognostic covariates. Results: 15170 out of 16703 pts in ESME had full IHC data allowing their classification as HR+HER2- (N=9922), HER2+ (N=2863), or HR-HER2- (N=2321) cases. Median FU and OS for the whole cohort are 4.05 yrs [95 CI: 3.98-4.12], and 3.1 yrs [95 CI: 3.03-3.18] respectively. In the adjusted multivariate analysis, year of MBC diagnosis, age at MBC, subtype (using HER2+ as reference), disease-free interval (DFI), visceral involvement, and number (nbr) of metastatic sites are significant OS predictors (table) although with low effect for the first item. Age at MBC, DFI, visceral involvement, and nbr of metastatic sites remained significant prognostic variables in subtypes. Year of diagnosis was no longer significant in HR+HER2- nor HR-HER2- cases (HR=0.997, p=0.71 and HR=0.997, p=0.84), while it was highly significant in HER2+ cases (HR=0.91, p<0.0001). Conclusions: Although OS of MBC has slightly improved over the past decade, this remains mostly confined to HER2+ cases, highlighting the need for new strategies for the luminal and triple negative populations. [Table: see text]

The role of taxanes in HR+ve/HER2-ve metastatic breast cancer (MBC) patients (pts) from adjuvant to metastatic setting in the clinical practice: Results from GIM13-AMBRA study.

1055 Background: The molecular subtypes of BC have individual patterns of behavior, prognosis and sensitivity to treatment, with subsequent implications for the choice of, or indeed role, for adjuvant (Adj) and metastatic chemotherapy (CHT). Taxanes (T) play a central role in chemotherapy for BC. However, previous studies have reported that T are relatively ineffective in patients with Luminal (HR+ve) BC compared with other subtypes. Aim of the present analysis is to describe the use of T in the clinical practice in Italy in HR+ve pts. Methods: AMBRA is a longitudinal cohort study, aiming to describe the choice of first and subsequent lines of treatment in HER2-ve MBC pts receiving at least one CHT (SABCS 2016, P5-15-07 & P5-14-09) in the years 2012-2015. For the present analysis, we focused on the use of T from the AdJ to the metastatic setting. Results: So far, 791/1500 pts have been registered into the study, 651 of them (82,3%) evaluable with HR+ MBC. Main characteristics are: Mean age 52 years; pN: UK 64 (9.8%) N+=405 (62.2%); Adj CHT=397 (61 %), mean DFI=96,99 months. T were used in 60.3% of the cases in the Adj setting, alone or in combination with other drugs, mainly anthracyclines (82.6%), with a mean DFI of 56.9 months. In the metastatic setting, across 1st to 3rd line, 460 pts (70.6%) received T, alone (49.7%) or in combination with Bevacizumab (38.2%), or other CHT drugs (11.9%). Details of the use of T in MBC are described in the table below. Conclusions: T have been used in more than 60% of cases of Luminal tumours in the Adj setting. In this population DFS is significantly shorter as compared with the non-T treated luminal population, as previously suggested by different Authors. Re-challenge with T is very frequent across different lines for MBC. Paclitaxel is the most used T, Docetaxel the less used and Nab-paclitaxel, labelled for MBC only, shows an increasing use from 1st-to 3rd line of treatment. [Table: see text]

Label retention and stem cell marker expression in the developing and adult prostate identifies basal and luminal epithelial stem cell subpopulations

BackgroundProstate cancer is the second most frequent cancer among males worldwide, and most patients with metastatic disease eventually develop therapy-resistant disease. Recent research has suggested the existence of cancer stem-like cells, and that such cells are behind the therapy resistance and progression.MethodsHere, we have taken advantage of the relatively quiescent nature of stem cells to identify the slow-cycling label-retaining stem cell (LRC) populations of the prostate gland. Mice were pulsed with bromodeoxyuridine (BrdU) during prostate organogenesis, and the LRC populations were then identified and characterized in 5-day-old and in 6-month-old adult animals using immunohistochemistry and immunofluorescence.ResultsQuantification of LRCs in the adult mouse prostate showed that epithelial LRCs were significantly more numerous in prostatic ducts (3.7 ± 0.47% SD) when compared to the proximal (1.4 ± 0.83%) and distal epithelium (0.48 ± 0.08%) of the secretory lobes. LRCs were identified in both the basal and epithelial cell layers of the prostate, and LRCs co-expressed several candidate stem cell markers in a developmental and duct/acini-specific manner, including Sca-1, TROP-2, CD133, CD44, c-kit, and the novel prostate progenitor marker cytokeratin-7. Importantly, a significant proportion of LRCs were localized in the luminal cell layer, the majority in ducts and the proximal prostate, that co-expressed high levels of androgen receptor in the adult prostate.ConclusionsOur results suggest that there are separate basal and luminal stem cell populations in the prostate, and they open up the possibility that androgen receptor-expressing luminal stem-like cells could function as cancer-initiating and relapse-responsible cells in prostate cancer.

Abstract P4-11-01: A genetic model of mammary gland fibrosis and cancer risk

Abstract Ductal cells of the mammary gland arise from mammary stem cells (MSC) and consist of estrogen receptor a negative (ER-) luminal cells and a small ER+ luminal population. Luminal cells are maintained by the proliferation of ER- luminal progenitor (LP) cells, which are expanded in basal subtype breast cancer. Human breast LP cells exhibit telomere DNA damage, which is associated with mammographic density and increased cancer risk. Short telomeres resulting from the DNA damage response correlate with aggressive breast cancer phenotypes. Telomeric repeat factor 2 (TRF2) binds to telomeres and protects them from the DNA damage response. TRF2 expression is reduced in most human breast cancers but the phenotypic advantages of this genetic alteration are unknown. To determine the effects of telomere DNA damage on mammary gland function and tumor phenotype, we deleted TRF2 expression in mammary gland epithelium. Mammary glands lacking TRF2 expression exhibited increased telomere DNA damage response, histopathological and functional degeneration, and prominent ductal fibrosis. MSC from TRF2 deficient glands were unable to completely regenerate mammary glands following transplantation to cleared fat pads. TRF2 deficient mammary tumors also demonstrated telomere DNA damage response but exhibited rapid onset and increased proliferation. Tumor derived LP cells underwent expansion but exhibited high telomere DNA response and failed to form tumors following transplantation. In contrast, the severely depleted MSC population was highly tumorigenic and maintained telomeres via the ALT mechanism. Telomere DNA damage response in mammary tumors resulted in p53 dependent ER+ cellular differentiation and sensitivity to anti-estrogen therapy. Our results provide a new in vivo model for exploring mechanisms of mammographic density, stem cell differentiation, cancer risk, and therapeutic sensitivity. Citation Format: Wu J, Crowe D. A genetic model of mammary gland fibrosis and cancer risk [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 P4-11-01.

Celecoxib Alters the Intestinal Microbiota and Metabolome in Association with Reducing Polyp Burden.

Treatment with celecoxib, a selective COX-2 inhibitor, reduces formation of premalignant adenomatous polyps in the gastrointestinal tracts of humans and mice. In addition to its chemopreventive activity, celecoxib can exhibit antimicrobial activity. Differing bacterial profiles have been found in feces from colon cancer patients compared with those of normal subjects. Moreover, preclinical studies suggest that bacteria can modulate intestinal tumorigenesis by secreting specific metabolites. In the current study, we determined whether celecoxib treatment altered the luminal microbiota and metabolome in association with reducing intestinal polyp burden in mice. Administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APC(Min/+) mice. Treatment with celecoxib also altered select luminal bacterial populations in both APC(Min/+) and wild-type mice, including decreased Lactobacillaceae and Bifidobacteriaceae as well as increased Coriobacteriaceae Metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites linked to carcinogenesis, including glucose, amino acids, nucleotides, and lipids. Ingenuity Pathway Analysis suggested that these changes in metabolites may contribute to reduced cell proliferation. To this end, we showed that celecoxib reduced cell proliferation in the base of normal appearing ileal and colonic crypts of APC(Min/+) mice. Consistent with this finding, lineage tracing indicated that celecoxib treatment reduced the rate at which Lgr5-positive stem cells gave rise to differentiated cell types in the crypts. Taken together, these results demonstrate that celecoxib alters the luminal microbiota and metabolome along with reducing epithelial cell proliferation in mice. We hypothesize that these actions contribute to its chemopreventive activity. Cancer Prev Res; 9(9); 721-31. ©2016 AACR.

Abstract LB-276: The identification and characterization of prostate adenocarcinoma tumor initiating cells

Abstract Primary prostate cancer is typically of luminal phenotype. However, little is known about the stem/progenitor properties of transformed luminal tumor cells as they fail to survive in culture. Using the organoid culture methodology, we show two distinct luminal progenitors in aggressive Pten/Tp53-null mouse model of prostate cancer. Not only did tumors contain previously described multipotent progenitors, but also a major population of committed luminal progenitors. The distinction between committed luminal and multipotent organoids was also evident in subcutaneous grafts as tumors of adenocarcinoma or multilineage histological phenotypes were observed, respectively. Moreover, using organoids we show that the self-renewing capacity of luminal-committed progenitors is a tumor-specific property, absent in benign luminal cells. Further, a significant fraction of luminal progenitors displayed resistance to in vivo castration as well as to androgen receptor inhibition ex vivo. Importantly, 3D organoid techniques have allowed us to relate our findings in humans as we can successfully grow similar luminal tumor populations from patient derived xenografts (PDXs) models of prostate cancer. In all, these data reveal two distinct luminal tumorigenic populations in mouse models of prostate cancer, providing insight into luminal tumor initiating cells in prostate cancer that can also influence response to therapy. Citation Format: Supreet Agarwal, Paul Hynes, Ross Lake, Lei Fang, Heather Tillman, Mike Beshiri, Keith Jansson, Wouter Karthaus, Philip Iaquinta, Charles Sawyers, Kathleen Kelly. The identification and characterization of prostate adenocarcinoma tumor initiating 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 LB-276.

Abstract 3672: Coordinated roles of CHD1 and SPOP in prostate cancer pathogenesis

Abstract Prostate cancer (PCa) is a clinically heterogeneous disease, consequently presenting challenges while designing efficacious therapeutic strategies across diverse patient cohorts. There is an increasing need to improve our understanding of the disease by classifying PCa into distinct molecular subtypes characterized by specific genomic alterations. To this end, loss of the gene encoding chromodomain helicase DNA binding protein 1 (CHD1) represents one of the largest classes of recurrent gene deletions detected exclusively in PCa, hallmarking up to 20% of characterized tumors. CHD1 is well known to facilitate the assembly of euchromatin during transcription. Therefore, we hypothesized that loss of CHD1 would induce a significant change in overall transcription and alter prostate cell fate, promoting tumorigenesis. To confer a high degree of physiological relevance to our studies, we utilized genetically engineered mouse (GEM) models harboring prostate-specific CHD1 deletions. Since loss of CHD1 has a significant tendency to co-occur with mutations in the SPOP gene, we generated GEM models that also co-express the SPOP F133V transgene. We derived organoid lines from GEM prostates to recapitulate prostate histology in a three dimensional matrix, which allowed us to track changes at the molecular level and overall phenotype as they relate to PCa development. We found that CHD1 loss associated strongly with the presence of an increased luminal cell population compared to wild type controls. Since luminal cells are recognized to be cells of origin in PCa, this observation is consistent with the notion that CHD1 deletion represents a pro-tumorigenic event. Additionally, our data indicates that co-occurrence of CHD1 deletions and SPOP mutations may confer a growth advantage to prostate cells. The overarching goal of this study is to define the coordinated role of CHD1 loss and SPOP mutations in PCa, highlight altered signaling pathways and phenotypes, and nominate potential therapeutic strategies for patients with this PCa subtype. Citation Format: Divya Vasudevan, Mirjam Blattner, Lesa D. Deonarine, Yu Chen, Mark A. Rubin, Christopher E. Barbieri. Coordinated roles of CHD1 and SPOP in prostate cancer pathogenesis. [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 3672.

Abstract 911: BRM loss promotes tumor progression through extracellular matrix remodeling and elevated mammary epithelial stem/progenitor activity

Abstract BRM expression is progressively lost in breast cancer and low levels are predictive of poor patient prognosis. We have previously shown that oncogenic downregulation of BRM promotes malignancy of mammary epithelial cells (MECs) through the CEBP/â-mediated induction of á5-integrin. BRM and BRG1 are necessary but mutually exclusive subunits of the SWI/SNF ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions to alter accessibility of specific genomic regions. To explore the physiological role of BRM in the normal mammary gland and in breast cancer, we studied BRM germline knockout mice (BRM KO). We first backcrossed BRM KO mice onto a clean genetic background and observed a 40% increase in animal size compared to wild type littermates. Histological examination of developing BRM KO mammary glands revealed enhanced ductal branching and a 60% increase in numbers of terminal end buds. Concordantly, immunohistochemistry for phospho-histone H3 exhibited significantly elevated proliferation of BRM KO MECs. Picrosirius red staining of mammary glands further demonstrated an accumulation of collagen around epithelial ducts in BRM KO mice that correlated with an upregulation of genes encoding the extracellular matrix (ECM) proteins Collagen-I and Fibronectin as well as the collagen remodeling enzymes Loxl1 and Loxl2 as assessed by quantitative PCR (qPCR). Subsequent flow cytometry analysis of mammary glands to sort luminal versus basal MEC populations demonstrated a preference for expansion of the basal lineage in BRM KO mice compared to controls. Basal MECs are in direct contact with the ECM and mammary stem cells are proposed to exist within this population. To investigate the possibility that BRM KO augments mammary stemness, we performed colony formation and limiting dilution transplantation assays, which served to confirm that the expanded basal population was indeed associated with elevated MEC stem/progenitor activity and also to verify the MEC dependence of these effects. To gain more mechanistic insight, we sorted luminal/basal MEC populations for qPCR analysis and found that MECs from BRM KO mice produce more abundant transcripts of genes associated with stemness. Our preliminary data now suggests a compensatory upregulation of BRG1 in BRM KO MECs and elevated expression of several known YAP/TAZ target genes involved in the control of cell proliferation. Finally, to describe a role for BRM in tumor progression, we examined the outcome of BRM KO in MMTV-NEU mice and observed more rapid tumor growth with a trend to higher tumor incidence and metastasis in mice lacking BRM. Taken together, our data indicate that SWI/SNF chromatin remodeling regulates YAP/TAZ control over MEC growth and a loss of BRM expression leads to BRG1-directed ECM remodeling, MEC proliferation and elevated mammary stemness, resulting in heightened mammary tumor aggression. Citation Format: Jason J. Northey, Laura Damiano, Valerie M. Weaver. BRM loss promotes tumor progression through extracellular matrix remodeling and elevated mammary epithelial stem/progenitor activity. [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 911.

Abstract LB-302: Celecoxib alters the intestinal microbiota and metabolome in association with reducing polyp burden

Abstract Introduction: Celecoxib is a chemopreventive agent that reduces adenomatous polyp number in the gastrointestinal tract of humans and mice and is thought to exert its effects exclusively through inhibition of COX-2. Bacteria have been shown to impact upon intestinal tumorigenesis in part by secretion of key metabolites. However, it is unknown whether celecoxib may be reducing polyp burden at least in part, through effects on the luminal microbiota or metabolome. This study tested whether celecoxib can alter the luminal microbiota and metabolome in association with reducing intestinal polyps in APCMin/+ mice, potentially providing a novel mechanism by which it exerts its chemopreventive effects. Methods: APCMin/+ and wild-type (WT) mice were co-housed for 2 weeks to normalize their microbiota, then individually housed and administered control or celecoxib-containing diet from 6 to 16 weeks of age. Feces were collected at 6, 11 and 16 weeks of age and ileal content and intestinal tissue (to quantify polyp burden) were collected at 16 weeks. Bacterial composition analysis on ileal content and feces was carried out using 16S rDNA sequencing and relative fecal metabolite levels were measured using UPLC/MS/MS and GC/MS. Results: As expected, administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APCMin/+ mice. Concomitant with this effect, drug administration altered the luminal bacterial populations, a shift that also occurred in WT mice. These changes included increased Coriobacteriaceae and decreased Lactobacillaceae and Bifidobacteriaceae. Subsequent metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites including glucose, amino acids, nucleotides and lipids. Ingenuity Pathway Analysis revealed that this change in metabolomic profile was associated with pathway perturbations that could contribute to reduced cell proliferation. Lastly, we showed that the observed changes were associated with a reduction in stem cell proliferation in normal intestinal crypts of APCMin/+ mice. Conclusions: These results demonstrate that celecoxib treatment profoundly altered the luminal microbiota and metabolome of mice and we hypothesize that these actions contribute to its chemopreventive activity. Further studies are warranted to confirm this hypothesis and assess whether our findings extend to humans and to other nonsteroidal anti-inflammatory drugs. Citation Format: David C. Montrose, Xi K. Zhou, Erin M. McNally, Erika Sue, Steven S. Gross, Nitai D. Leve, Edward D. Karoly, Lilan Ling, Eric G. Pamer, Andrew J. Dannenberg. Celecoxib alters the intestinal microbiota and metabolome in association with reducing polyp burden. [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 LB-302.

Abstract 3308: Bisphenol A treatment induces hyperplasia in primary and stem cell-generated mammary glands from pregnant mice

Abstract Breast cancer is a commonly diagnosed cancer during pregnancy. However, how endogenous and exogenous endocrine factors may contribute to the development of pregnancy-associated mammary tumorigenesis is not clear. There is growing evidence that mammary stem cells (MaSCs) may initiate neoplastic transformation when dysregulated in mouse models. We investigated the effect of the environmental endocrine disruptor bisphenol A (BPA) on mouse mammary gland morphology, epithelial cell composition, pre-neoplastic lesions, and the regenerative function of MaSCs. Pregnant FVB mice with GFP transgene on Day E8.5 were implanted with osmotic pumps that constantly release BPA at 0, 25 or 250 ng/kg/day for 28 days and the mice were euthanized one month after weaning. In agreement with the literature, we observed an abnormality of the morphology of the mammary gland after BPA treatment characterized by higher duct density and abnormal secondary and tertiary branching. Quantification of percent hyperplastic mammary ducts in H&E-stained tissue slides revealed a significant increase of ducts with hyperplastic lesions after BPA treatment, particularly with the low dose. To investigate the effects of BPA treatment on MaSCs, we used enzyme digestion to isolate the CD24hi/CD49f+ luminal epithelial cells (also termed as colony forming cell or CFC) and the CD24+/CD49fhi basal epithelial cells (also termed as mammary repopulating unit or MRU) from mammary gland tissues by FACS and found no significant difference in percent of luminal or basal cell population after BPA treatment. Because the basal cells are enriched with MaSCs that can form mammospheres in suspension culture and subsequently form solid 3D organoids when cultured in Matrigel, we transplanted the solid 3D organoids into cleared mammary fat pads of syngeneic FVB mice and immune-compromised nude mice to examine how BPA treatment might alter MaSC function. Significantly, similar to the results from the primary mammary glands, the regenerated mammary glands by MaSCs from mice treated with the low dose of BPA showed increased duct density, secondary and tertiary branching, and a significantly greater number of hyperplastic lesions. Taken together, our study demonstrated that BPA exposure at very low dose could induce pre-neoplastic lesions in the mammary gland of pregnant mice, apparently by directly targeting MaSCs, and implicates BPA as an exogenous endocrine factor that may promote pregnancy-associated mammary tumorigenesis. Citation Format: Hakim Bouamar, Fuchuang Zhang, Xiang Gu, Qiaoxiang Dong, Lu-Zhe Sun. Bisphenol A treatment induces hyperplasia in primary and stem cell-generated mammary glands from pregnant mice. [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 3308.

Abstract 514: Effect of radiation therapy on breast epithelial cells in BRCA1/2 mutation carriers

Abstract Women carrying BRCA1 and BRCA2 mutations have significantly elevated risk of developing breast and ovarian cancers. BRCA1-associated breast cancer likely originates from progenitors of the luminal epithelial lineage. Recent studies indicate that radiation therapy (RT) for BRCA1 cancer patients is associated with lower incidence of developing subsequent ipsilateral breast cancer. In the current study, we analyzed tumor-free breast tissue procured via prophylactic bilateral mastectomy from three BRCA1 and one BRCA2 mutation carriers, who had been previously treated with RT for unilateral breast cancers. Freshly isolated breast cells from the irradiated and non-irradiated breasts of the same individuals were subjected to flow cytometry, using established cell-surface markers. Two out of the three BRCA1 carriers and the BRCA2 carrier exhibited significantly diminished luminal cell population in the irradiated breast versus the non-irradiated side. The remaining BRCA1 carrier showed similar cell abundance between the two sides. Our finding suggests that prior RT could result in preferential depletion of the luminal epithelial compartment and thus reduced incidence of BRCA1-associated breast cancer. Further investigation may inform development of RT-based preventive approaches aimed at specifically decimating the source of BRCA1-associated tumors. Citation Format: Huai-Chin Chiang, Richard Elledge, Paula Larson, Ismail Jatoi, Rong Li, Yanfen Hu. Effect of radiation therapy on breast epithelial cells in BRCA1/2 mutation carriers. [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 514.

Abstract LB-279: N-cadherin promotes castration-resistant prostate cancer progression by enhancing stem cell properties of prostate cancer cells

Abstract New generation of anti-androgen treatments such as enzalutamide and abiraterone are improving survival of men with advanced and metastatic prostate cancers, but resistance to these inevitably arises. The molecular mechanisms governing the emergence of treatment resistance in castration resistant prostate cancer (CRPC) patients are not well understood. Recent experience suggests that tumor regeneration from castration-resistant stem-like cells induce resistance to hormonal therapy. Therefore, elucidating novel targets essential for driving stem-like activity is critical to prevent and defeat CRPC. It has been shown that normal and cancer stem cells exploit normal development process of epithelial-mesenchymal transition (EMT) to survive and metastasize, and that EMT confer stem cell properties to more differentiated cancer cell progeny in breast and other cancers. However, it is unclear if EMT is linked with stem cells in normal and malignant prostate. We previously showed that N-cadherin, a marker of EMT, promotes CRPC progression and that there was a correlation between N-cadherin expression and the expression of stem cell associated markers. This leads to hypothesis that N-cadherin may promote CRPC by enhancing tumorige esis/tumor initiating properties of cancer cells in a castrate environment. N-cadherin may confer tumor initiating or stem-like properties to prostate cancer cells perhaps uniquely or specific to in a castrate environment. Here, we verified that EMT is linked to stem cells in both normal prostate and CRPC. We further demonstrate that N-cadherin is involved in increased stem cell properties in normal mouse and human prostates from clinical samples in an androgen-deprived environment. Castration elevates the expression of N-cadherin and stem cell marker Trop2, accompanied with enhanced stem cell properties of luminal population in normal mouse prostates. N-cadherin upregulates stem cell properties in prostate cancer cells in the absence of androgen and CRPC tumors. N-cadherin-positive cells from CRPC tumors behave as stem cells. Overexpression of N-cadherin enhances ALDH activity in a panel of prostate cancer cell lines. Our findings reveal an important role of N-cadherin in prostate cancer and stem cells, and provide useful information of EMT-related biomarkers for preventing and developing efficient therapeutics to combat the treatment resistance to next-generation AR antagonists. Citation Format: Shu Lin, Tanya Stonyanova, Evelyn Kono, Jaclyn Matsuura, Naoko Kobayashi, Joyce Yamashiro, Dai Hong, Owen N. Witte, Andrew S. Goldstein, Robert E. Reiter. N-cadherin promotes castration-resistant prostate cancer progression by enhancing stem cell properties of prostate cancer 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 LB-279.

Transcriptome profiling links the intrinsic properties of human prostate basal cells to prostate cancer aggressiveness

ABSTRACTGenome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations reveals the unexpected findings that basal cells preferentially express gene categories associated with stem cells, neurogenesis, and rRNA biogenesis. Importantly, the basal cell gene expression profile is enriched in clinically advanced, anaplastic, castration-resistant, and metastatic prostate cancers.

Prostate epithelial cell of origin determines cancer differentiation state in an organoid transformation assay

The cell of origin for prostate cancer remains a subject of debate. Genetically engineered mouse models have demonstrated that both basal and luminal cells can serve as cells of origin for prostate cancer. Using a human prostate regeneration and transformation assay, our group previously demonstrated that basal cells can serve as efficient targets for transformation. Recently, a subpopulation of multipotent human luminal cells defined by CD26 expression that retains progenitor activity in a defined organoid culture was identified. We transduced primary human prostate basal and luminal cells with lentiviruses expressing c-Myc and activated AKT1 (myristoylated AKT1 or myrAKT1) to mimic theMYCamplification andPTENloss commonly detected in human prostate cancer. These cells were propagated in organoid culture before being transplanted into immunodeficient mice. We found that c-Myc/myrAKT1-transduced luminal xenografts exhibited histological features of well-differentiated acinar adenocarcinoma, with strong androgen receptor (AR) and prostate-specific antigen (PSA) expression. In contrast, c-Myc/myrAKT1-transduced basal xenografts were histologically more aggressive, with a loss of acinar structures and low/absent AR and PSA expression. Our findings imply that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types subjected to the same oncogenic insults. This study provides a platform for the functional evaluation of oncogenes in basal and luminal epithelial populations of the human prostate. Tumors derived in this fashion with defined genetics can be used in the preclinical development of targeted therapeutics.

Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer

The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

FOXA1 expression affects the proliferation activity of luminal breast cancer stem cell populations.

The expression of estrogen receptor is the key in most breast cancers (BC) and binding of estrogen receptor to the genome correlates to Forkhead protein (FOXA1) expression. We herein assessed the correlation between the cancer stem cell (CSC) population and FOXA1 expression in luminal BC. We established luminal BC cells derived from metastatic pleural effusion and analyzed the potency of CSC and related factors with established luminal BC cell lines. We also confirmed that mammosphere cultures have an increased aldehyde dehydrogenase‐positive population, which is one of the CSC markers, compared with adherent culture cells. Using a quantitative PCR analysis, we found that mammosphere forming cells showed a higher expression of FOXA1 and stemness‐related genes compared with adherent culture cells. Furthermore, the growth activity and colony‐forming activity of 4‐hydroxytamoxifen‐treated BC cells were inhibited in a mammosphere assay. Interestingly, 4‐hydroxytamoxifen‐resistant cells had significantly increased FOXA1 gene expression levels. Finally, we established short hairpin RNA of FOXA1 (shFOXA1) MCF‐7 cells and investigated the relationship between self‐renewal potential and FOXA1 expression. As a result, we found no significant difference in the number of mammospheres but decreased colony formation in shFOXA1 MCF‐7 cells compared with control. These results suggest that the expression of FOXA1 appears to be involved in the proliferation of immature BC cells rather than the induction of stemness‐related genes and self‐renewal potency of CSCs.