Expression Of Basal Markers Research Articles

Are Basal-Like and Non-Basal-Like Triple-Negative Breast Cancers Really Different?

Objective Triple-negative breast cancer (TNBC) accounts for 15–25% of breast cancers. It is increasingly recognized that TNBC is a motley disease. TNBC and basal-like (BL) subtype are different molecular classes of breast cancer with a high degree of overlap. However, a smaller fraction lacks the expression of basal markers in spite of being TNBC and is called non-basal-like (NBL). The aim of this study is to assess the clinicopathological features in TNBC and compare its BL and NBL subtypes. Material and Methods. A total of 200 subjects fulfilling the inclusion criteria of study were identified from the electronic medical records of institution. The tumor sections of subjects were immunohistochemically stained for basal markers, namely, 34βE12, c-Kit, and EGFR, in order to differentiate between BL and NBL subtypes. Comprehensive data were assembled from subjects' clinical records. The features of TNBC and their associations with the two subtypes were assessed using statistical analyses. Results TNBC constituted 22% of all breast cancers. The family history of cancer was observed to be significantly associated with stage (p=0.013). The proportions of BL and NBL subtypes were equal. Of all parameters compared between two subtypes, only lymphovascular invasion was found to have statistically significant difference (p=0.019). Though no statistical significant difference between overall survival (OS) and disease-free survival (DFS) of two subgroups was found, BL subtype has slightly shorter DFS and OS compared to NBL. Conclusion Both BL and NBL subtypes occur in equal proportions; hence, basalness and triple negativity are not synonyms. Though BL and NBL are prognostically similar, BL subtype shows a trend towards slightly shorter DFS and OS compared to NBL.

Platform Effects on Regeneration by Pulmonary Basal Cells as Evaluated by Single-Cell RNA Sequencing

SUMMARYCell-based therapies have shown promise for treating myriad chronic pulmonary diseases through direct application of epithelial progenitors or by way of engineered tissue grafts or whole organs. To elucidate environmental effects on epithelial regenerative outcomes in vitro, here, we isolate and culture a population of pharmacologically expanded basal cells (peBCs) from rat tracheas. At peak basal marker expression, we simultaneously split peBCs into four in vitro platforms: organoid, air-liquid interface (ALI), engineered trachea, and engineered lung. Following differentiation, these samples are evaluated using single-cell RNA sequencing (scRNA-seq) and computational pipelines are developed to compare samples both globally and at the population level. A sample of native rat tracheal epithelium is also evaluated by scRNA-seq as a control for engineered epithelium. Overall, this work identifies platform-specific effects that support the use of engineered models to achieve the most physiologic differential outcomes in pulmonary epithelial regenerative applications.

Co-expression of HLA-I loci improved prognostication in HER2+ breast cancers.

The underlying basis for cancer immune evasion is important for effective immunotherapy and prognosis in breast cancers. Human leucocyte antigens (HLA)-I comprising three classical antigens (HLA-A, -B and -C) is mandatory for anti-tumor immunity. Its loss occurred frequently in many cancers resulting in effective immune evasion. Most studies examined HLA-I as a whole. Alterations in specific locus could have different clinical ramifications. Hence, we evaluated the expression of the three HLA-I loci in a large cohort of breast cancers. Low expression of HLA-A, -B and -C were found in 71.1%, 66.3%, and 60.2% of the cases. Low and high expression in all loci was found in 48.3% and 17.9% of the cases respectively. The remaining showed high expression in one or two loci. Cases with all HLA high expression (all HLA high) was frequent in the ER-HER2- (27.4%) and ER-HER2+ (23.1%) cases and was associated with characteristic pathologic features related to these tumor (higher grade, necrosis, high tumor infiltrating lymphocyte (TIL), pT stage, low hormonal receptor, high basal marker expression) (p ≤ 0.019). Interestingly, in HER2+ cancers, only cases with all HLA high and high TIL showed significantly better survival. In node positive cancers, concordant high HLA expression in primary tumors and nodal metastases was favorable prognostically (DFS: HR = 0.741, p < 0.001; BCSS: HR = 0.699, p = 0.003). The data suggested an important clinical value of a combined analysis on the co-expression HLA-I status in both primary and metastatic tumors. This could be a potential additional key component to be incorporated into TIL evaluation for improved prognostication.

VDR in salivary gland homeostasis and cancer

The vitamin D receptor (VDR) and its ligand 1,25(OH)2D3 (1,25D) impact differentiation and exert anti-tumor effects in many tissues, but its role in salivary gland has yet to be defined. Using immunohistochemistry (IHC), we have detected strong VDR expression in murine and human salivary gland ducts. Compared to normal gland, VDR protein expression was retained in differentiated human pleomorphic adenoma (PA) but was undetectable in undifferentiated PA and in carcinomas, suggesting deregulation of VDR during salivary cancer progression. To gain insight into the potential role of VDR in salivary cancer, we assessed the effects of vitamin D in vivo and in vitro. Despite the presence of VDR in salivary gland, chronic dietary vitamin D restriction did not alter morphology of the salivary epithelium in C57/Bl6 mice. The localization of VDR in ductal epithelium prompted us to examine the effects of 1,25D in an established cell line (mSGc) derived from normal murine submandibular gland (SMG). This previously characterized cell line consists of multiple stem, progenitor and differentiated cell types as determined by mutually exclusive cellular expression of basal, ductal and myoepithelial markers. We demonstrated VDR expression and regulation of VDR target genes Vdr and Postn by 1,25D in mSGc, indicating functional ligand-mediated transcriptional activity. The effect of VDR signaling on epithelial differentiation markers was assessed by qPCR and IHC in mSGc cells treated with 1,25D. We found that 1,25D reduced mRNA expression of the basal cell progenitor marker keratin 5 (K5) and increased expression of the differentiated ductal cell marker keratin 7 (K7). Further, we found that 1,25D significantly decreased the number of proliferating cells, including proliferating K5+ cells. Characterization of cell cycle by Muse cytometry indicated 1,25D treatment decreased cells in S, G2, and M phase. The inhibition of K5+ cell proliferation by 1,25D is of particular interest because K5+ basal cells contribute to a wide variety of salivary tumor types. Our studies suggest that 1,25D alters cancer-relevant progenitor and differentiation markers in the salivary gland.

Immunohistochemical Expression of Basal Cell and Urothelial Markers (p63, Uroplakin III) in Non-Neoplastic and Neoplastic Canine Prostate Tissue

Immunohistochemical Expression of Basal Cell and Urothelial Markers (p63, Uroplakin III) in Non-Neoplastic and Neoplastic Canine Prostate Tissue

Y-27632 preserves epidermal integrity in a human skin organ-culture (hSOC) system by regulating AKT and ERK signaling pathways

BackgroundThe human skin organ culture (hSOC) developed a century ago has been widely used to study various aspects of human skin development, differentiation, function, disease as well as skin appendages biology, however, maintaining the integrity of epidermal structure in long-term culture, has remained a challenge. ObjectivesHere we tried to establish a culture system using supplemented William’s E medium in the presence of a ROCK inhibitor Y-27632 to maintain epidermal architecture in the long-term hSOC and to investigate the underlying mechanisms. MethodsHuman breast skins, cut into 5 mm × 5 mm pieces, were cultured in supplemented William’s E medium in the presence of 30μM Y-27632. The cultured skin tissues were collected at different time points for analysis of epidermal cell proliferation and differentiation by real time qRT-PCR and immunofluorescence (IF) staining. The keratinocyte suspension assay and in vivo treatment of Y-27632 on mouse were also carried out to study that the regulation of Y-27632 on keratinocyte proliferation and differentiation. ResultsWe found Y-27632 not only enhanced both basal cell proliferation and expression of suprabasal cell differentiation markers, but also maintained the balance of keratinocyte proliferation and differentiation through activation of AKT pathways on one hand and inhibition of ERK pathways on the other hand. The AKT inhibitor MK-2206 blocked the epidermal preservation effect of Y-27632, while the MEK/ERK inhibitor U0126 enhanced the preservation of epidermal structure in the hSOC. ConclusionsY-227632 can maintain skin epidermal integrity through regulation of AKT and ERK activity in the hSOC.

Stability of αB-crystallin gene expression in canine mammary gland neoplasms. Should it be considered as circulating tumor cell genetic marker?

αB-crystallin is a member of a small family of thermal shock proteins that protects cells from stress. Because of lack of its expression in peripheral blood leukocytes, it was proposed as a molecular marker of circulating tumor cells in canine mammary gland tumors. The aim of the present study was to determine if αB-crystallin shows stability of expression, what is the requirement for this type of marker. It was also assessed whether there is co-expression of αB-crystallin with the basal marker, cytokeratin 17. For this purpose, samples of various types of canine mammary gland tumors of epithelial origin, were selected. Using RT-qPCR, we have found αB-crystallin and cytokeratin 17 co-expression in benign and malignant canine mammary gland tumors. It has been demonstrated that the expression of αB-crystallin in tested neoplastic samples is not stable in comparison to the control group. Furthermore αB-crystallin overor down- expression was associated witch the same cytokeratin 17 pattern. αB-crystallin can be a marker of circulating tumor cells in the bloodstream, but for cancers in which basal marker expression occurs and thus not universal for all cancers originating from the mammary gland tissue.

Clinicopathological and epidemiological significance of breast cancer subtype reclassification based on p53 immunohistochemical expression

TP53 mutations are common in breast cancer and are typically associated with more aggressive tumor characteristics, but little is known about the clinicopathological and epidemiological relevance of p53 protein expression, a TP53 mutation surrogate, in breast cancer subtypes. In this study of 7226 Chinese women with invasive breast cancer, we defined breast cancer subtypes using immunohistochemical (IHC) measures of hormone receptors and HER2 in conjunction with histologic grade. p53 expression status was then used to further stratify subtypes into p53-positive and p53-negative. Odds ratios (ORs) and 95% confidence intervals (CIs) in case-only logistic regression analyses were used to examine heterogeneity across different subtypes. The frequency of p53 protein expression varied by breast cancer subtype, being lowest in the luminal A-like and highest in the triple-negative and HER2-enriched subtypes (P-value < 0.01). In luminal A-like and B-like/HER2-negative subtypes, p53 positivity was associated with early-onset tumors, high grade, high proliferative index, and basal marker (CK5/6 and EGFR) expression. Further, compared with luminal A-like/p53-negative patients, A-like/p53-positive patients were more likely to be parous [adjusted OR parous vs. nulliparous = 2.67 (1.60, 4.51); P-value < 0.01] and to have breastfed [adjusted OR ever vs. never = 1.38 (1.03, 1.85); P-value = 0.03]. p53 positivity was not associated with examined clinical and risk factors in other tumor subtypes. Overall, these findings suggest that p53 expression, which is readily available in many settings, can be used to identify phenotypes of luminal A-like breast cancer with distinct clinical and epidemiological implications.

Molecular Characterization of Neuroendocrine-like Bladder Cancer.

Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping studies have found that 5% to 15% of muscle-invasive bladder cancer (MIBC) have transcriptomic patterns consistent with NE bladder cancer in the absence of NE histology. The clinical implications of this NE-like subtype have not been explored in depth. Transcriptome-wide expression profiles were generated for MIBC collected from 7 institutions and clinical-use of Decipher Bladder. Using unsupervised clustering, we generated a clustering solution on a prospective training cohort (PTC; n = 175), developed single-sample classifiers to predict NE tumors, and evaluated the resultant models on a testing radical cystectomy (RC) cohort (n = 225). A random forest model was finalized and applied to 5 validation cohorts (n = 1302). Uni- and multivariable survival analyses were used to characterize clinical outcomes. In the training cohort (PTC), hierarchical clustering using an 84-gene panel showed a cluster of 8 patients (4.6%) with highly heterogeneous expression of NE markers in the absence of basal or luminal marker expression. NE-like tumors were identified in 1% to 6.6% of cases in validation cohorts. Patients with NE-like tumors had significantly worse 1-year progression-free survival (65% NE-like vs. 82% overall; P = 0.046) and, after adjusting for clinical and pathologic factors, had a 6.4-fold increased risk of all-cause mortality (P = 0.001). IHC confirmed the neuronal character of these tumors. A single-patient classifier was developed that identifies patients with histologic urothelial cancer harboring a NE transcriptomic profile. These tumors represent a high-risk subgroup of MIBC, which may require different treatment.

Basal-Like Breast Cancer; Clinicopathological, Molecular, and Prognostic Features

Background: Molecular classification of breast tumors has identified the basal-like subtype, with high heterogeneity and very poor prognosis. These tumors are mainly triple negative, characterized by the expression of basal markers CK5/6 and EGFR. In this study, we sought to investigate the features, outcome, and therapeutic modalities of basal-like breast cancers (BLBC).Methods: We retrospectively identified 90 BLBC patients diagnosed at the Department of Surgical Oncology of Salah Azaiez Institute between January 2009 and December 2013. Results: The mean age of our patients was 50 years, and 15.5% had a family history of breast cancer. The mean tumor size was 43.8 mm. Histological examination revealed invasive ductal carcinoma in 88.9% of the cases, metaplastic carcinoma in 5.6%, and medullary carcinoma and adenoid cystic carcinoma in 2.2%. BLBC was most often associated with a high tumor grade (55.3% had a grade 3 tumor) and a high Ki-67 proliferative index. Vascular invasion was found in 31.1% of the cases. Regarding lymph node involvement, 42.9% had positive lymph nodes and 7.9% featured distant metastases. Surgical treatment was provided for 85 patients. It consisted of conservative surgery in 40 cases and radical surgery in 45 cases. Neoadjuvant chemotherapy was administrated to 23 patients, with a 13% complete pathologic response. The rates of overall survival and disease-free survival at 3 years for localized BLBC were 74.4% and 75.9%, respectively. Conclusion: BLBCs are aggressive tumors associated with poor prognosis. Thus, to identify novel prognostic factors and therapeutic targets, prospective studies should investigate the epidemiological and evolutive profile of these tumors.

P63 at the Crossroads between Stemness and Metastasis in Breast Cancer.

After lung cancer, breast cancer (BC) is the most frequent cause of cancer death among women, worldwide. Although advances in screening approaches and targeted therapeutic agents have decreased BC incidence and mortality, over the past five years, triple-negative breast cancer (TNBC) remains the breast cancer subtype that displays the worst prognosis, mainly due to the lack of clinically actionable targets. Genetic and molecular profiling has unveiled the high intrinsic heterogeneity of TNBC, with the basal-like molecular subtypes representing the most diffuse TNBC subtypes, characterized by the expression of basal epithelial markers, such as the transcription factor p63. In this review, we will provide a broad picture on the physiological role of p63, in maintaining the basal epithelial identity, as well as its involvement in breast cancer progression, emphasizing its relevance in tumor cell invasion and stemness.

Induced Pluripotent Stem Cell‐Derived Basal Cells Provide A Novel Source of Multilineage Airway Epithelial Cells

IntroductionGene‐corrected autologous cells provide one avenue for treating respiratory diseases, such as Cystic Fibrosis and Primary Ciliary Dyskinesia that harbor an underlying genetic defect. Basal cells are critical stem cells in the airways with the capacity to generate all cells of the respiratory epithelium. Use of these cells is limited due to inherent difficulties in gene editing and expansion in vitro. Efficient generation of basal cells from iPSCs has the potential to generate enough cells for both autologous cell therapy and for in vitro disease modelling. Building on our airway differentiation protocol, we have derived a strategy for the specification, isolation and expansion of iPSC‐derived basal cells (iBasal) improving the efficiency of generating respiratory epithelium from iPSC.MethodsiPSC are cultured using a modified differentiation protocol pushing the formation of lung progenitor cells sorted on day 17 of differentiation for CD47hiCD26lo cells. These cells express transcription factor NKx2.1 and can be cultured in defined serum‐free media in the absence of wnt signaling. Over 4 passages we evaluated; 1) expression of basal cell markers, 2) clonality by a limited dilution clonogenic assay and by spheroid formation and 3) stem cell potential in spheroid and air‐liquid interface differentiation assays. Gene expression was compared to that of primary endogenous basal cells by bulk and single cell RNAseq, and validated by qPCR.ResultsUnder our defined culture conditions, iBasal can be derived from NKx2.1 expressing lung progenitor cells. They are clonogenic forming colonies expressing cytokeratin 5 (krt5), p63 and integrin alpha 6 (IGTA6), acquiring more mature basal cell marker, nerve growth factor receptor (NGFR), expression after passaging when cultured on NIH3T3‐J2 feeders or in defined media. iBasal have the capacity for differentiation into a functional, tight junction forming epithelium containing both mucus secreting and motile multiciliated cells at the air liquid interface. In addition, they can form and maintain as spheroids which can also differentiate to generate airway epithelial cells. iBasal can be passaged and maintain a basal cell phenotype similar to that of paired passaged human airway basal cells. Single cell sequence data on iBasal and paired primary and cultured basal cells will provide the ultimate validation of iBasal.ConclusionWe have generated iBasal from four independent iPSC lines with functional properties akin to that of primary basal cells. iBasal, therefore, provide the opportunity to generate sufficient functional and gene‐corrected autologous basal cells to develop patient specific, high throughput screening platforms overcoming the current limitations.Support or Funding InformationCystic Fibrosis Foundation FIRTH17XX0, FIRTH15XX1, NIH:NHLBI R01 HL139828‐01, Hastings FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mammary Epithelial Cell Lineage Changes During Cow's Life.

Milk production is highly dependent on the optimal development of the mammary epithelium. It is therefore essential to better understand mammary epithelial cell growth and maintenance from the related epithelial lineage during the animal life. Here, we characterized the epithelial lineage at puberty, lactation and dry-off in bovine using the cell surface markers CD49f, CD24, and CD10. The pubertal period was characterized by a high proportion of CD49fpos cells corresponding to various epithelial subpopulations, notably the CD24pos subpopulations. The proportion of CD49fpos cells was weaker during lactation and dry-off, and CD24pos cells were relatively few. Of note, the (sub)population profile at dry-off appeared close to that during lactation. Using a targeted gene approach, we associated specific genes with epithelial subpopulations, their expression level varying, or not, according to physiological stages. Caseins were only expressed in the CD49fmedCD24neg subpopulation. Basal marker genes (keratin(KRT)5, KRT14 and αSMA) were found in the CD49fhighCD24neg subpopulations. Luminal gene markers (KRT7, KRT8 and KRT19, CDH1 and the PRLR) were expressed in the CD49flowCD24neg subpopulation. The CD49flowCD24pos subpopulation, only abundant at puberty, expressed luminal gene markers and KI67 at high level. In contrast to others, the CD49fhighCD24pos cells accounted for a small proportion of total cells, decreasing from puberty to dry-off. They were characterized by expression of luminal and basal gene markers and low KI67 level. Interestingly, this subpopulation showed a remarkable stability of gene expression profile throughout physiological stages and bear the hallmark of quiescence that designate them as the potential bovine mammary stem cells.

TGF-β Upregulated Mitochondria Mass through the SMAD2/3→C/EBPβ→PRMT1 Signal Pathway in Primary Human Lung Fibroblasts.

Tissue remodeling of subepithelial mesenchymal cells is a major pathologic condition of chronic obstructive pulmonary disease and asthma. Fibroblasts contribute to fibrotic events and inflammation in both airway diseases. Recent mechanistic studies established a link between mitochondrial dysfunction or aberrant biogenesis leading to tissue remodeling of the airway wall in asthma. Protein arginine methyltransferase-1 (PRMT1) participated in airway wall remodeling in pulmonary inflammation. This study investigated the mechanism by which PRMT1 regulates mitochondrial mass in primary human airway wall fibroblasts. Fibroblasts from control or asthma patients were stimulated with TGF-β for up to 48 h, and the signaling pathways controlling PRMT1 expression and mitochondrial mass were analyzed. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI-1. The SMAD2/3 pathway was blocked by SB203580 and C/EBPβ by small interference RNA treatment. The data obtained from unstimulated cells showed a significantly higher basal expression of PRMT1 and mitochondrial markers in asthmatic compared with control fibroblasts. In all cells, TGF-β significantly increased the expression of PRMT1 through SMAD2/3 and C/EBPβ. Subsequently, PRMT1 upregulated the expression of the mitochondria regulators PGC-1α and heat shock protein 60. Both the inhibition of the SAMD2/3 pathway or PRMT1 attenuated TGF-β-induced mitochondrial mass and C/EBPβ and α-SMA expression. These findings suggest that the signaling sequence controlling mitochondria in primary human lung fibroblasts is as follows: TGF-β→SMAD2/3→C/EBPβ→PRMT1→PGC-1α. Therefore, PRMT1 and C/EBPβ present a novel therapeutic and diagnostic target for airway wall remodeling in chronic lung diseases.

The urothelial cell line UROtsa transformed by arsenite and cadmium display basal characteristics associated with muscle invasive urothelial cancers.

Muscle invasive urothelial carcinomas are divided into various molecular subtypes with basal and luminal subtypes being the prominent ones. The basal muscle-invasive urothelial carcinomas are generally more aggressive at presentation and significantly enriched with squamous features. Our laboratory has developed an in-vitro model of urothelial cancer by transforming the immortalized cell line UROtsa with arsenite (As3+) and cadmium (Cd2+). In this study, we characterized the tumors formed by these transformed cell lines as more basal-like based on their gene expression patterns with increased expression of KRT1, KRT5, KRT6, KRT14, KRT16, KRT17 and CD44. In addition, histological examination of these tumor transplants showed squamous features enriched in basal muscle invasive urothelial carcinomas. The expression of these genes increased in the transformed cell lines as well as in the urospheres, which are putative cancer initiating cells/stem cells derived from the cell lines. There was also increased expression of these genes in the urospheres derived from the parent UROtsa cell line. Thus, our data shows that the As3+ and Cd2+-transformed cell lines and their derived tumor transplants have gene expression profiles similar to the basal subtype of muscle invasive bladder carcinomas with tumors having enriched squamous features. The increased expression of basal markers in the urospheres suggests that stem cells may be involved in the development of squamous differentiation seen in some of the muscle invasive bladder carcinomas.

P53 Silencing and Mammosphere Formation in Breast Cancer Cells Harbouring P53 Gain-of-Function Mutations Trigger Epithelial-Mesenchymal Transition

Background: Mutations in p53 gene are observed in ∼50% of all human cancers. In breast cancer alone, 12%-32% of luminal, 84% of basal-like and 75% of HER-2 expressing tumors have apparent p53 mutations. Tumor cells undergo epithelial-mesenchymal transition (EMT) to metastasise from primary sites to form secondary tumors at distant regions of the body. EMT is a complex biologic phenomenon which governs the transition of cancer cells with epithelial characteristics to mesenchymal traits, gaining new properties such as aggressiveness and invasiveness. Recent studies revealed that mutations in the p53 gene can give rise to alternate functional phenotypes leading to tumor initiation and progression. Aim: The aim of this study is to develop a robust human breast cancer cellular model to investigate p53 gain-of-function (GOF) mutations and EMT as well as evaluating the EMT phenotype associated with these mutations. Methods: Two breast cancer cell lines, namely MDA-MB-468 and HCC38 carrying the R273H and R273L missense mutations, respectively, were subjected to p53 knockdown using shRNA directed against p53 gene through lentiviral vector transduction. The transduced cells were then harvested for Western blotting to evaluate the protein expression of EMT markers which includes E-cadherin, SNAIL, ZEB1 and vimentin compared with the nontransduced control cells. Subsequently, both cell lines were subjected to mammosphere generation and redifferentiation to determine the basal expression of the EMT markers. Results: Silencing of p53 using siRNA in MDA-MB-468 and HCC38 downregulated E-cadherin expressions but upregulated vimentin levels. Furthermore, E-cadherin levels reduced significantly after conversion from adherent parental cells to mammospheres, but rebound upon redifferentiation. Conversely, vimentin was upregulated in mammospheres as compared with the parental and redifferentiated groups. Conclusion: p53 knockdown in breast cancer cells harboring R273H and R273L mutations favor vimentin expression but not E-cadherin, suggesting that p53-GOF mutants are involved in EMT and the development of metastatic tumors. The mammosphere model accurately recapitulates cell plasticity between epithelial and mesenchymal states, as evidenced by the expression of mesenchymal cell markers in the mammospheres, and epithelial cell markers in adherent and redifferentiated cells.

Development of xeno-free epithelial differentiation media for adherent, non-expanded adipose stromal vascular cell cultures

IntroductionReconstruction of respiratory epithelium is critical for the fabrication of bioengineered airway implants. Epithelial differentiation is typically achieved using bovine pituitary extract (BPE). Due to the xenogenic nature and undefined composition of BPE, an alternative for human clinical applications, devoid of BPE, must be developed. The goal of this study was to develop two different BPE-free media, with and without select pituitary hormone (PH), which could initiate epithelial differentiation for use in human implantation. MethodsThe ability of the two BPE-free media to initiate epithelial differentiation of adherent, non-expanded stromal-vascular cells grown on porcine small intestinal submucosa was compared to traditional BPE-containing media (M1). Nanostring® was used to measure differences in gene expression of stemness (MSC), basal cell (basal), and ciliated markers (muco-cil), and staining was performed support the gene data. ResultsCompared to baseline, both BPE-free media upregulated epithelial and stemness genes, however this was to a lower degree than BPE-containing media. In general, the expression of basal cell markers (COL17A1, DSG3, ITGA6, KRT6A, LOXL2) and secreted mucous proteins (PLUNC, MUC5B, SCGB2A1) was upregulated. The gene expression of ciliated markers C9orf24, TUBA3 and DNCL2B but not of the key transcription factor for cilagenesis FOXJ1 were upregulated, indicating that mucus-secreting cell differentiation occurs more rapidly than ciliogenesis. The ability of the adherent stromal vascular cells to upregulate gene expression of both epithelial and stemness markers suggests maintenance of the self-renewal capacity of undifferentiated and/or basal cell-like cells contributing to proliferation and ensuring a persisting source of cells for regenerative medicine applications. ConclusionThis study provides the initial step to defining a BPE-free epithelial differentiation medium for clinical translation. Thus, either of the proposed BPE-free medium are viable alternatives to BPE-containing medium for partial epithelial differentiation for human translational applications.

Amyloid Precursor Protein Is Associated with Aggressive Behavior in Nonluminal Breast Cancers.

β-amyloid precursor protein (APP), a potential target for Alzheimer's disease treatment, has recently been shown to take part in carcinogenesis. Increased APP promotes migration, survival, and proliferation in breast cancer cell lines. We examined the clinical value of APP in breast cancers. A comprehensive examination of clinicopathological features related to APP expression in a large cohort of breast cancers and the corresponding metastatic lymph nodes was performed. APP expression and its prognostic impact in different breast cancer subtypes were examined. APP was highly expressed in nonluminal breast cancers and correlated with features associated with nonluminal breast cancers (including higher grade, the presence of necrosis, and higher proliferative index, growth factor receptor, and basal marker expression). Multivariate Cox hazard analysis demonstrated that APP was an independent adverse prognostic factor of disease-free survival (DFS; hazard ratio [HR], 2.090; p = .013; 95% confidence interval [CI], 1.165-3.748) and breast cancer-specific survival (BCSS; HR, 2.631; p = .002; 95% CI, 1.408-4.915) in the nonluminal group. The independent prognostic impact was also seen in triple negative breast cancers. Interestingly, a higher expression of APP was found in nodal metastasis compared with primary tumor. Such APP upregulation was correlated with further distal metastasis and poorer outcome (DFS: log-rank, 12.848; p < .001; BCSS: log-rank, 13.947; p < .001). Our findings provided evidence of oncogenic roles of APP in clinical breast cancers. Patients with positive APP expression, particularly those with APP upregulation in lymph node metastases, may require vigilant monitoring of their disease and more aggressive therapy. β-amyloid precursor protein (APP), a potential target for Alzheimer's disease, has recently been implicated in oncogenesis. Here, evidence of its roles in clinical breast cancers is provided. Positive APP expression was found to be an independent prognostic factor in nonluminal cancers, particularly triple negative breast cancers (TNBCs). Interestingly, a higher APP in nodal metastases was associated with distal metastases. TNBCs are heterogeneous and currently have no available target therapy. APP could have therapeutic potential and be used to define the more aggressive cases in TNBCs. Current prognostic analysis is based on primary tumor. The present data suggest that investigation of nodal metastases could provide additional prognostic value.

Abstract A040: Targeting the prostate cancer stem cell niche with the STAT3 inhibitor galiellalactone

Abstract Introduction and Objectives: Cancer stem-like cells (CSCs) represent a small subpopulation of largely quiescent cells that reside within tumors. Several studies have demonstrated that this population is more resistant to current therapies and is therefore directly responsible for tumor recurrence. Recent evidence suggests that the transcription factor STAT3 is crucial to the survival of stem cells in prostate cancer (PCa). Thus, inhibition of activated STAT3 (pSTAT3) is a valid strategy to selectively target PCa CSCs. We have previously shown that pSTAT3 blockade, by galielallactone (GL), not only reduces proliferation and induces apoptosis of prostate cancer cells in vitro, but it also inhibits the growth of prostate tumors and the metastatic spread to regional and distal lymph nodes, in vivo. Here we performed experiments aimed at studying the effect of the STAT3 inhibitor, GL, on PCa CSCs, as a promising therapeutic approach for prostate cancer patients. Materials and Methods: The expression of stem, basal, and luminal cell surface markers (CD133/CD44/CD24) was analyzed by FACS on DU145 and PC3 cells following treatment with GL. DU145 cells were sorted, based on the expression of the stem cell surface marker CD133, and then plated for further analysis. Expression of pSTAT3 was studied by IHC, on sorted cells, and the effect of GL on clonogenic recovery was determined. Furthermore, the effect of GL on sphere formation efficiency was studied on the CD133 population. The compound galiellalactone was obtained from Glactone Pharma (Sweden). Results: Our results show that the CSCs population (CD133+/CD44+) expresses high levels of activated pSTAT3 compared to the CD44+/CD24+ cell population. Treatment with GL decreased the number of CSCs in DU145 cells after 48h, while the number of CD44+/CD24+ cells was not significantly affected. Importantly, treatment with GL had no effect on CD133+/CD44+ or CD44+/CD24+ populations in PC3 cells, which do not express pSTAT3. Treatment with GL significantly reduced the ability of CSCs to form colonies and to generate prostate-derived spheres. Interestingly, the CSCs population (CD133+) was more sensitive to GL in terms of colony- and sphere-forming efficiency compared to the CD133- population. Conclusion: This study demonstrates that the STAT3 inhibitor galiellalactone can specifically target the prostate cancer stem-like cell population in vitro, implying that pSTAT3 inhibition by GL could represent a valid therapeutic strategy to antagonize CSCs in human prostate cancer. Future experiments will be aimed at validating these data and relating the clonogenic inhibition to the effects of pSTAT3 blockade by GL on tumor initiation by prostate cancer stem-like cells in vivo. Citation Format: Giacomo Canesin, Anne T. Collins, Rebecka Hellsten, Norman J. Maitland, Anders Bjartell. Targeting the prostate cancer stem cell niche with the STAT3 inhibitor galiellalactone [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 A040.

CD147 expression is associated with poor overall survival in chemotherapy treated triple-negative breast cancer

AimsIn breast cancer models, the functional roles of CD147 in proliferation, invasion and treatment resistance have been widely reported. However, there are only a few studies examining the clinicopathological correlation...