Expression Of Basal Markers Research Articles

Hyaluronan synthase 2 is an adverse prognostic marker in androgen receptor-negative breast cancer

AimsThe important role of hyaluronan synthase 2 (HAS2), an isozyme responsible for hyaluronan synthesis, in cancer has been increasingly recognised. However, only a few studies with inconsistent results have been...

Abstract P6-08-02: Developing in vitro models of ductal carcinoma in situ from primary tissue

Abstract BACKGROUND: Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive aggressive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. MATERIALS AND METHODS: From 2/2014 to 4/2015, patients with percutaneous core needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Under supervision of the surgical pathologist, fresh tissue measuring between 5-15 mm in length was taken from lumpectomy or mastectomy specimens. Tissue was divided such that half was mechanically and enzymatically dissociated and then cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and the second half, known as the "mirror image" remained as part of the clinical specimen. RESULTS: Of 49 consented patients, mean age was 59 ± 10 years. 7 were excluded due to final pathology not consistent with DCIS: 4 upstaged to invasive ductal cancer, 2 had microinvasion and 1 showed pleomorphic lobular carcinoma in situ. Of the remaining 42, 9 were failures: 5 tissues were not received in lab and 4 cases were received, but no cells grew in culture. Of the remaining 33 cases of DCIS, 70% (n=23) and 27% (n=9) were nuclear grade 2 and 3 respectively. 91% (n=30) were ER-positive, with H-score ranging between 4 and 300. 19 (58%) were expanded in cell culture for up to two months in culture, and 14 were frozen immediately after mechanical dissociation for future growth. The 19 cell cultures could be cryopreserved and expanded. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal cells and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the "conditionally reprogrammed" proliferative state and when conditioned media and ROCK inhibitor were withdrawn. When grown to 100% confluency, the cultures appear to organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. CONCLUSION: Primary cultures of DCIS derived directly from patient tissues may serve as in vitro models for the study of DCIS. Citation Format: McAuliffe PF, Brown DD, Oesterreich S, Lee AV, Johnson RR, McGuire KP, Davidson NE, Brufsky AM, Dabbs DJ. Developing in vitro models of ductal carcinoma in situ from primary tissue. [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 P6-08-02.

Molecular Classification of Breast Cancer Patients Using Formalin-fixed Paraffin-embedded Derived RNA Samples

The use of archival formalin-fixed paraffin-embedded (FFPE) material to analyse gene expression is limited by the low quality of extracted RNA. In this paper, we utilised an RNA based assay to quantify expression of luminal and basal markers, together with ERBB2 probes, in FFPE archival tissue from 2009 to 2010, all of which had clinical and therapeutic information of more than 5 years. Receptor status of the patients was characterised using the QuantiGene® Plex assay with 100% concordance to immunohistochemical (IHC) and fluorescence in situ hybridisation (FISH) results. A panel of molecular markers known to classify luminal and basal tumours were used and correlated with receptor status of the tumours. As expected, the triple negative breast cancer (TNBC) samples were classified as basal and oestrogen receptor (ER) positive cases as luminal. In summary, the QuantiGene® Plex technology provides a platform to quantitate novel panels of biomarkers on archival material. Moreover, multiplex analysis allows the use of minimal amounts of material providing an opportunity to utilise laser micro-dissected material. FFPE tissue samples are an invaluable resource for retrospective studies to interrogate current novel biomarkers, particularly to generate disease free survival and overall survival graphs to measure predictive value using well annotated retrospective samples with full clinical and pharmacological outcomes.

Disrupted in schizophrenia 1 (DISC1) L100P mutants have impaired activity-dependent plasticity in vivo and in vitro.

Major neuropsychiatric disorders are genetically complex but share overlapping etiology. Mice mutant for rare, highly penetrant risk variants can be useful in dissecting the molecular mechanisms involved. The gene disrupted in schizophrenia 1 (DISC1) has been associated with increased risk for neuropsychiatric conditions. Mice mutant for Disc1 display morphological, functional and behavioral deficits that are consistent with impairments observed across these disorders. Here we report that Disc1 L100P mutants are less able to reorganize cortical circuitry in response to stimulation in vivo. Molecular analysis reveals that the mutants have a reduced expression of PSD95 and pCREB in visual cortex and fail to adjust expression of such markers in response to altered stimulation. In vitro analysis shows that mutants have impaired functional reorganization of cortical neurons in response to selected forms of neuronal stimulation, but there is no altered basal expression of synaptic markers. These findings suggest that DISC1 has a critical role in the reorganization of cortical plasticity and that this phenotype becomes evident only under challenge, even at early postnatal stages. This result may represent an important etiological mechanism in the emergence of neuropsychiatric disorders.

Heterogeneity of luminal breast cancer characterised by immunohistochemical expression of basal markers.

Background:Luminal A breast cancer defined as hormone receptor positive and human epidermal growth factor receptor 2 (HER2) negative is known to be heterogeneous. Previous study showed that luminal A tumours with the expression of basal markers ((cytokeratin (CK) 5 or CK5/6) or epidermal growth factor receptor (EGFR)) were associated with poorer prognosis compared with those that stained negative for basal markers. Prompted by this study, we assessed whether tumour characteristics and risk factors differed by basal marker status within luminal A tumours.Methods:We pooled 5040 luminal A cases defined by immunohistochemistry (4490 basal-negative ((CK5 (or CK5/6))− and EGFR−) and 550 basal-positive ((CK5 (or CK5/6+)) or EGFR+)) from eight studies participating in the Breast Cancer Association Consortium. Case–case comparison was performed using unconditional logistic regression.Results:Tumour characteristics and risk factors did not vary significantly by the expression of basal markers, although results suggested that basal-positive luminal tumours tended to be smaller and node negative, and were more common in women with a positive family history and lower body mass index.Conclusions:Most established breast cancer risk factors were similar in basal-positive and basal-negative luminal A tumours. The non-significant but suggestive differences in tumour features and family history warrant further investigations.

Phenotypic Modulation and Neuroprotective Effects of Olfactory Ensheathing Cells: a Promising Tool for Cell Therapy.

Olfactory Ensheathing Cells (OECs), exhibiting phenotypic characteristics of both astrocytes and Schwann Cells, show peculiar plasticity. In vitro, OECs promote axonal growth, while in vivo they promote remyelination of damaged axons. We decided to further investigate OEC potential for regeneration and functional recovery of the damaged Central Nervous System (CNS). To study OEC antigen modulation, OECs prepared from postnatal mouse olfactory bulbs were grown in different culture conditions: standard or serum-free media with/without Growth Factors (GFs) and analyzed for different neural specific markers. OEC functional characterizations were also achieved. Resistance of OECs to the neurotoxin 6-hydroxydopamine (6-OHDA) was analyzed by evaluating apoptosis and death. OEC neuroprotective properties were investigated by in vitro co-cultures or by addition of OEC conditioned medium to the neuroblastoma SH-SY5Y cells exposed to 6-OHDA. We observed: 1) modification of OEC morphology, reduced cell survival and marker expression in serum-free medium; 2) GF addition to serum-free medium condition influenced positively survival and restored basal marker expression; 3) no OEC apoptosis after a prolonged exposition to 6-OHDA; 4) a clear OEC neuroprotective tendency, albeit non statistically significant, on 6-OHDA treated SH-SY5Y cells. These peculiar properties of OECs might render them potential clinical agents able to support injured CNS.

The Potential Role of Hedgehog Signaling in the Luminal/Basal Phenotype of Breast Epithelia and in Breast Cancer Invasion and Metastasis.

The epithelium of the lactiferous ducts in the breast is comprised of luminal epithelial cells and underlying basal myoepithelial cells. The regulation of cell fate and transit of cells between these two cell types remains poorly understood. This relationship becomes of greater importance when studying the subtypes of epithelial breast carcinoma, which are categorized according to their expression of luminal or basal markers. The epithelial mesenchymal transition (EMT) is a pivotal event in tumor invasion. It is important to understand mechanisms that regulate this process, which bears relation to the normal dynamic of epithelial/basal phenotype regulation in the mammary gland. Understanding this process could provide answers for the regulation of EMT in breast cancer, and thereby identify potential targets for therapy. Evidence points towards a role for hedgehog signaling in breast tissue homeostasis and also in mammary neoplasia. This review examines our current understanding of role of the hedgehog-signaling (Hh) pathway in breast epithelial cells both during breast development and homeostasis and to assess the potential misappropriation of Hh signals in breast neoplasia, cancer stem cells and tumor metastasis via EMT.

Developing in vitro models of human ductal carcinoma in situ from primary tissue explants.

Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive comprehensive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. Patients with percutaneous core-needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Fresh tissue was taken from lumpectomy or mastectomy specimens, mechanically and enzymatically dissociated, cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and characterized by immunocytochemistry. Out of 33 DCIS cases, 58% (19) were expanded for up to 2 months in culture, and 42% (14) were frozen immediately after mechanical dissociation for future growth. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the "conditionally reprogrammed" proliferative state and after conditioned media and ROCK inhibitor withdrawal. When grown to 100 % confluency, the cultures organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. Primary cultures of DCIS derived directly from patient tissues can be generated and may serve as in vitro models for the study of DCIS.

Abstract 1861: Estrogen modulation of fibroblast growth factor signaling in non-small cell lung cancer

Abstract The estrogen signaling pathway induces proliferation in non-small cell lung cancer (NSCLC) and represents a novel therapeutic target for lung cancer. Estrogen receptor β (ERβ) is the predominant ER isoform in lung cancer and we have previously shown that high cytoplasmic ERβ combined with low progesterone receptor (PR) expression is a poor prognostic marker for NSCLC patients. To further elucidate the biological differences between ERβ high/PR low versus ERβ low/PR high expressing lung tumors, a mRNA microarray analysis using the Illumina HT-12 V4 Bead Chip platform was performed using RNA extracted from 64 cases. 165 genes were differentially expressed between these two groups at P<001. Fibroblast growth factor receptor 1 (FGFR1) was a highly over-expressed gene associated with ERβ high tumors (2.1-fold, P<0.004), while the decoy receptor for FGFR ligands, fibroblast growth factor receptor like-1 (FGFR5), was down-regulated (0.54-fold, P<0.0001). Ingenuity Pathway analysis revealed a network among the 165 differentially expressed genes that linked FGFR1 and FGFR5 to the ER. To analyze cross-talk between the FGF and E2 pathways, we determined the FGF/FGFR profile in a panel of three FGFR1 amplified and five non-amplified NSCLC cell lines. FGF2, 3, 10, and 19 were the most highly secreted FGFs in the cell line panel. FGFR1 amplified cells secreted statistically more FGF3, 6 and 10 compared to non-amplified cells. β-estradiol (E2) induced secretion of FGF2 in a cell line without FGFR1 amplification. FGFR3 and 5 were ubiquitously expressed across all cell lines while FGFR1, 2 and 4 expression was higher in amplified cell lines. There was no relationship between ERβ protein expression and FGFR1 amplification status or FGFR1-5 protein expression. Because FGF signaling has been shown to increase stem cell-like phenotypes in breast cancer, we determined basal and E2-stimulated expression of the stem cell markers, SOX2 and OCT4. Basal SOX2 and OCT4 were more highly expressed in FGFR1 amplified cells compared to non-amplified cells. SOX2 and OCT4 protein expression were increased by E2 treatment in FGFR1 non-amplified cells, with lesser effects in FGFR1 amplified H520 cells. SOX2 expression was also down-regulated in lung sections from mice treated with a combination of the anti-estrogen fulvestrant and the aromatase inhibitor anastrozole. Finally, we have shown that in FGFR1 non-amplified cells, the anti-proliferative effect of a pan-FGFR inhibitor, AZD4547, can be enhanced in the presence of the anti-estrogen fulvestrant. These results suggest that there is an interaction between the estrogen and FGF signaling pathways in lung cancer and provides a rationale for combination treatment of NSCLC tumors. Further characterization of the molecular types of NSCLC that will benefit from joint therapy is warranted. This work was supported by P50 CA090440. Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Marjorie Romkes, Lisa Koodie, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Estrogen modulation of fibroblast growth factor signaling in non-small cell lung cancer. [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 1861. doi:10.1158/1538-7445.AM2015-1861

Abstract 5570: Heterogeneity of luminal breast cancer characterized by immunohistochemical expression of basal markers

Abstract Data from epidemiologic studies demonstrated that some established breast cancer risk factors such as parity and obesity had differential effect on luminal (estrogen receptor (ER) positive or progesterone receptor (PR) positive, and HER2 negative) and CBP subtypes (ER-, PR-, HER2- and express basal markers [cytokeratin 5/6 (CK5/6) (or CK5) and/or the epidermal growth factor receptor (EGFR)], known as the core basal phenotype), suggesting that luminal and CBP subtypes may have different underlying etiology. Recent data suggest that a subset of ER+ tumors with preexisting CK5+ cells may be resistant to hormone therapies and represent a clinically unique entity. In addition, luminal tumors expressing basal markers (luminal-basal) were associated with poorer prognosis compared to luminal tumors negative for basal markers. To assess whether luminal-basal tumors represent a distinct disease entity from an etiologic perspective, we pooled individual data for 6,112 invasive breast cancer cases contributed by five studies participating in the Breast Cancer Association Consortium (BCAC), with risk factor information and ER, PR, HER2, and basal marker data. We used unconditional logistic regression to evaluate relationships between risk factors and tumor subtypes by comparing luminal-basal tumors (ER+ or PR+, HER2-, CK5/6+ and/or EGFR+) to pure luminal A tumors (ER+ or PR+, HER2-, CK5/6-, EGFR-) in case-only analyses and to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between risk factors and specific tumor subtypes in 3 population-based studies including up to 4,732 cases and 13,242 controls. In case-only analyses, we found that postmenopausal cases with luminal-basal tumors were more likely to be parous (ORparous vs. nulliparous = 1.72; 95% CI = 1.06-2.78; P = 0.03), have higher number of full term births (ORtrend = 1.33; 95% CI = 1.07-1.66; P = 0.01), and lower frequency of obesity (ORBMI≥ 30 kg/m2 vs. <25 kg/m2 = 0.62; 95% CI = 0.40-0.95; P = 0.03) compared to cases with pure luminal A tumors. In case-control analysis, obesity, particularly postmenopausal obesity, was significantly associated with decreased risk of luminal-basal tumors (ORBMI≥ 30 kg/m2 vs. <25 kg/m2 = 0.57; 95% CI = 0.35-0.93; P = 0.02). In contrast, postmenopausal obesity was associated with increased risk of pure luminal A tumors, and premenopausal obesity was associated with increased risk of CBP tumors. In conclusion, our study suggests that luminal-basal cancers are more frequent among lean and parous postmenopausal women than pure luminal A cancers. Future studies with larger number of subjects and detailed annotation of subtype and risk factor information are needed to validate our findings and to more accurately assess etiologic and clinical heterogeneity within luminal A tumors. Citation Format: Hyuna Sung, Jenny Chang-Claude, Jonine Figueroa, Heli Nevanlinna, Graham G. Giles, Angelia Cox, Simon S. Cross, Melissa C. Southey, Catriona McLean, Manjeet K. Bolla, Mitul Shah, Alison M. Dunning, Joe Dennis, Qin Wang, Kyriaki Michailidou, Douglas F. Easton, Paul D. P. Pharoah, Mark E. Sherman, Montserrat Garcia-Closas, Xiaohong R. Yang. Heterogeneity of luminal breast cancer characterized by immunohistochemical expression of basal markers. [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 5570. doi:10.1158/1538-7445.AM2015-5570

Rapid creation of skin substitutes from human skin cells and biomimetic nanofibers for acute full-thickness wound repair

Creation of functional skin substitutes within a clinically acceptable time window is essential for timely repair and management of large wounds such as extensive burns. The aim of this study was to investigate the possibility of fabricating skin substitutes via a bottom-up nanofiber-enabled cell assembly approach and using such substitutes for full-thickness wound repair in nude mice. Following a layer-by-layer (L-b-L) manner, human primary skin cells (fibroblasts and keratinocytes) were rapidly assembled together with electrospun polycaprolactone (PCL)/collagen (3:1, w/w; 8%, w/v) nanofibers into 3D constructs, in which fibroblasts and keratinocytes were located in the bottom and upper portion respectively. Following culture, the constructs developed into a skin-like structure with expression of basal keratinocyte markers and deposition of new matrix while exhibiting good mechanical strength (as high as 4.0MPa by 14 days). Treatment of the full-thickness wounds created on the back of nude mice with various grafts (acellular nanofiber meshes, dermal substitutes, skin substitutes and autografts) revealed that 14-day-cultured skin substitutes facilitated a rapid wound closure with complete epithelialization comparable to autografts. Taken together, skin-like substitutes can be formed by L-b-L assembling human skin cells and biomimetic nanofibers and they are effective to heal acute full-thickness wounds in nude mice.

Short Communication: Low Expression of Activation and Inhibitory Molecules on NK Cells and CD4(+) T Cells Is Associated with Viral Control.

Chronic HIV-1 infection induces severe immune alterations, including hyperactivation, exhaustion, and apoptosis. In fact, viral control has been associated with low frequencies of these processes. Here, we evaluated the expression of activation and inhibitory molecules on natural killer (NK) and CD4(+) T cells and plasma levels of proinflammatory cytokines in individuals exhibiting viral control: a cohort of HIV-1-exposed-seronegative individuals (HESN) and a cohort of HIV controllers. There was lower expression of CD69, LAG-3, PD-1, and TIM-3 in both cohorts when compared to a low-risk population or HIV progressors. In addition, HIV controllers exhibited lower plasma levels of proinflamatory molecules TNF-α and IP-10. These findings suggest that individuals exhibiting viral control have lower basal expression of markers associated with cellular activation and particularly immune exhaustion.

Different Growth Patterns of Canine Prostatic Carcinoma Suggests Different Models of Tumor-Initiating Cells.

Controversies remain regarding the cell type from which human prostate cancer originates, and many attempts have been made to identify the cellular origin of canine prostate cancer but without definitive proof. This study aims to evaluate the expression of luminal (androgen receptor [AR], cytokeratin [CK]8/18) and basal (CK14, CK5) cell markers in different histologic subtypes of canine prostatic carcinoma (PC) and to suggest the most likely tumor-initiating cells. Normal prostates (n = 8) were characterized by AR+CK8/18+ luminal cells and few CK5+ basal cells, while CK14 was absent. Similar pattern was observed in all 35 prostates with benign prostatic hyperplasia, except few scattered CK14+ basal cells in 13 samples (37.14%). AR was localized in the nucleus of both normal and hyperplastic cells. In 34 samples of PC, the following growth patterns were identified: cribriform (44.12%), solid (32.35%), small acinar/ductal (20.59%), and micropapillary (2.94%). Most PCs expressed AR and CK8/18, while CK5 and CK14 expression was observed in 25% and 20% of cases, respectively. AR revealed a variable intracellular distribution, both nuclear and cytoplasmic. Solid PC was characterized by an undifferentiated or aberrant phenotype with a reduced expression of AR and CK8/18, increased number of CK14+ cells, and 7 antigen expression patterns. This study demonstrated a predominance of differentiated luminal cell types in canine prostatic tumors, although the role of basal cells in prostate carcinogenesis should also be considered. Moreover, few scattered CK5+ cells in AR+CK8/18+ tumors identified the existence of intermediate cells, from which neoplastic transformation may alternatively commence.

Clinicopathological characteristics of triple negative breast cancer at a tertiary care hospital in India.

Triple-negative breast cancer (TNBC), characterized by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, is typically associated with a poor prognosis. The majority of TNBCs show the expression of basal markers on gene expression profiling and most authors accept TNBC as basal-like (BL) breast cancer. However, a smaller fraction lacks a BL phenotype despite being TNBC. The literature is silent on non-basal-like (NBL) type of TNBC. The present study was aimed at defining behavioral differences between BL and NBL phenotypes. i) Identify the TNBCs and categorize them into BL and NBL breast cancer. ii) Examine the behavioral differences between two subtypes. iii) Observe the pattern of treatment failure among TNBCs. All TNBC cases during January 2009-December 2010 were retrieved. The subjects fitting the inclusion criteria of study were differentiated into BL and NBL phenotypes using surrogate immunohistochemistry with three basal markers 34βE12, c-Kit and EGFR as per the algorithm defined by Nielsen et al. The detailed data of subjects were collated from clinical records. The comparison of clinicopathological features between two subgroups was done using statistical analyses. The pattern of treatment failure along with its association with prognostic factors was assessed. TNBC constituted 18% of breast cancer cases considered in the study. The BL and NBL subtypes accounted for 81% and 19% respectively of the TNBC group. No statistically significant association was seen between prognostic parameters and two phenotypes. Among patients with treatment failure, 19% were with BL and 15% were with NBL phenotype. The mean disease free survival (DFS) in groups BL and NBL was 30.0 and 37.9 months respectively, while mean overall survival (OS) was 31.93 and 38.5 months respectively. Treatment failure was significantly associated with stage (p=.023) among prognostic factors. Disease stage at presentation is an important prognostic factor influencing the treatment failure and survival among TNBCs. Increasing tumor size is related to lymph node positivity. BL tumors have a more aggressive clinical course than that of NBL as shown by shorter DFS and OS, despite having no statistically significant difference between prognostic parameters. New therapeutic alternatives should be explored for patients with this subtype of breast cancer.

Anatomical localization of progenitor cells in human breast tissue reveals enrichment of uncommitted cells within immature lobules.

IntroductionLineage tracing studies in mice have revealed the localization and existence of lineage-restricted mammary epithelial progenitor cells that functionally contribute to expansive growth during puberty and differentiation during pregnancy. However, extensive anatomical differences between mouse and human mammary tissues preclude the direct translation of rodent findings to the human breast. Therefore, here we characterize the mammary progenitor cell hierarchy and identify the anatomic location of progenitor cells within human breast tissues.MethodsMammary epithelial cells (MECs) were isolated from disease-free reduction mammoplasty tissues and assayed for stem/progenitor activity in vitro and in vivo. MECs were sorted and evaluated for growth on collagen and expression of lineages markers. Breast lobules were microdissected and individually characterized based on lineage markers and steroid receptor expression to identify the anatomic location of progenitor cells. Spanning-tree progression analysis of density-normalized events (SPADE) was used to identify the cellular hierarchy of MECs within lobules from high-dimensional cytometry data.ResultsIntegrating multiple assays for progenitor activity, we identified the presence of luminal alveolar and basal ductal progenitors. Further, we show that Type I lobules of the human breast were the least mature, demonstrating an unrestricted pattern of expression of luminal and basal lineage markers. Consistent with this, SPADE analysis revealed that immature lobules were enriched for basal progenitor cells, while mature lobules consisted of increased hierarchal complexity of cells within the luminal lineages.ConclusionsThese results reveal underlying differences in the human breast epithelial hierarchy and suggest that with increasing glandular maturity, the epithelial hierarchy also becomes more complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0453-3) contains supplementary material, which is available to authorized users.

Abstract 3047: Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells

Abstract Tumor initiating subpopulations of cancer cells, also known as cancer progenitor or cancer stem cells (CSCs) were recently identified and characterized in prostate cancer. Emerging evidence suggests that hypoxia-inducible factor 1 alpha (HIF1A) contributes to CSC maintenance and influences the PI3K/AKT/mTOR axis, a pathway which is frequently altered in prostate cancer. In our study, we assessed the effects of hypoxia on PI3K/AKT/mTOR signaling in a subset of basal prostate cancer stem cells in a murine model of prostate cancer. First, we successfully isolated Lin-/Sca-1+/CD49f+ basal stem cell subpopulations from primary prostate tumors of TRAMP (transgenic adenocarcinoma of mouse prostate) mice. We observed a consistent decrease in S6 phosphorylation within the CSC subpopulation, in agreement with an mTOR pathway inhibition. However, AKT phosphorylation in the CSC subpopulation was simultaneously elevated, thus prompting further investigations. To analyze the molecular pathways leading to this deregulation, we used a cell line model and isolated Sca-1+/CD49f+ CSC-like subpopulations from the murine prostate cancer cell line TRAMP-C1 and CD44+/CD49f+ CSC-like subpopulations from the androgen independent human prostate cancer cell line DU145. We used sphere formation assays and expression of Notch 1 and Oct 3/4 to confirm the stem and progenitor cell properties of the sorted subpopulations. We could confirm the decrease in mTOR/S6 signaling and identify the upregulation of AKT through IRS-1 mediated feedback loop in prostate CSC-like cells under hypoxic conditions. This AKT/mTOR deregulation was promptly reversed in normoxia, suggestive of the involvement of hypoxia inducible factors. We found elevated HIF1A protein levels as well as increased target gene expression in CSC subpopulations in vitro and in vivo. Further, lentiviral knockdown of HIF1A restored AKT/mTOR activity in CSC-like cells. We could show that CSC-like prostate cancer cells with elevated expression of basal stem cell markers, such as CD44, Sca-1 or CD49f, show a hypoxic deactivation of feedback inhibition on AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway via HIF1A may be critical for cancer stem cell quiescence and maintenance, slowing down cancer stem cell metabolism and growth via mTOR and promoting their survival via AKT signaling. Citation Format: Maximilian Marhold, Erwin Tomasich, Zuzana Pernicová, Radek Fedr, Karel Soucek, Andreas Spittler, Michael Krainer, Peter Horak. Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2014-3047

The basal phenotype as a clinically relevant indicator of trastuzumab resistance in HER2+ breast cancer.

154 Background: Trastuzumab (Herceptin) resistance remains a clinical challenge but the mechanism is not well understood. Recently, studies have identified a subset of Her2+ breast cancer called basal HER2 that expresses basal genes. We investigated the effect of basal gene expression on Herceptin response in HER2+ breast cancer cell lines and on prognosis in HER2+ breast cancer patients. Methods: Non-basal (BT474, SKBR3) and basal (HCC1569, HCC1954, JIMT-1) HER2+ cell lines were chosen based on basal cytokeratin expression. Cell proliferation was assessed after treatment with vehicle, Herceptin (H), Paclitaxel (P), H+P, Akt Inhibitor (AI), and H+AI. HER2 signaling was examined using immunoblotting of p-Akt and p-ERK. Because breast cancer stem cells (BCSC) are linked to basal breast tumors and treatment resistance, we assessed BCSC activity using mammosphere formation and aldehyde dehydrogenase (ALDH) positivity. Immunohistochemical staining of HER2+ breast cancers for basal markers CK5/6, CK14, and EGFR was correlated with clinicopathologic features and survival in 88 patients with Stage 1-3 HER2+ breast cancer treated with Herceptin. Results: Basal HER2 cells were resistant to Herceptin compared to non-basal HER2 cells but this resistance was overcome by Akt inhibition. Immunoblotting showed that non-basal HER2 cells had decreased p-Akt after Herceptin treatment which was not seen in basal HER2 cells. There was no difference in p-ERK levels after Herceptin therapy in all cell lines. Basal HER2 cells had increased mammosphere formation and ALDH positivity suggesting higher stem cell activity compared to non-basal HER2 cells. Of the HER2+ patients, 33/88 (37.5%) expressed at least one basal marker. Basal Her2 tumors were associated with higher grade (p = 0.04) and more ER/PR negativity (p < 0.01). CK14 expression correlated with worse overall survival by log-rank test (p = 0.02), while EGFR showed a similar trend (p = 0.06). Conclusions: Basal HER2 breast cancer cell lines have Herceptin resistance which may be due to constitutively active Akt signaling and increased stem cell activity. Clinically, basal marker expression predicts Herceptin resistance and worse outcomes in HER2+ breast cancer patients.

FOXA1 deletion in luminal epithelium causes prostatic hyperplasia and alteration of differentiated phenotype

The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1loxp/loxp mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1loxp/loxp prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.

P63 is more sensitive and specific than 34βE12 to differentiate adenocarcinoma of prostate from cancer mimickers.

Prostate cancer is the world's leading cause of cancer and the second cause of cancer-related death in men after lung cancer. Differentiation of prostate adenocarcinoma from benign prostate lesions and hyperplasia sometimes cannot be done on the basis of morphologic findings. Considering the fact that in the prostate adenocarcinoma there is no basal cell layer, basal cell markers can help to differentiate prostate adenocarcinoma from cancer mimickers. We studied 98 prostate biopsy blocks (40 adenocarcinoma and 58 benign lesions) for basal cell marker expression. p63 and 34βE12 were negative in all prostate adenocarcinoma specimens, but all benign prostate hyperplasia and high grade intraepithelial neoplasia cases expressed them. Basal cell markers can help to distinguish prostate adenocarcinoma from cancer mimickers.

Association of basal marker expression with outcome and trastuzumab resistance in HER2-positive breast cancer.

608 Background: Herceptin resistance is a significant challenge in the treatment of Her2-positive (Her2+) breast cancer. A subset of Her2+ breast cancers are known to express basal genes (basal Her...