Cancer Stem Cell Markers CD44 Research Articles

Infection with Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Human Cholangiocytes.

Recent reports suggest that the East Asian liver fluke infection, caused by Opisthorchis viverrini, which is implicated in opisthorchiasis-associated cholangiocarcinoma, serves as a reservoir of Helicobacter pylori. The opisthorchiasis-affected cholangiocytes that line the intrahepatic biliary tract are considered to be the cell of origin of this malignancy. Here, we investigated interactions in vitro among human cholangiocytes, Helicobacter pylori strain NCTC 11637, and the congeneric bacillus, Helicobacter bilis. Exposure to increasing numbers of H. pylori at 0, 1, 10, 100 bacilli per cholangiocyte of the H69 cell line induced phenotypic changes including the profusion of thread-like filopodia and a loss of cell-cell contact, in a dose-dependent fashion. In parallel, following exposure to H. pylori, changes were evident in levels of mRNA expression of epithelial to mesenchymal transition (EMT)-encoding factors including snail, slug, vimentin, matrix metalloprotease, zinc finger E-box-binding homeobox, and the cancer stem cell marker CD44. Analysis to quantify cellular proliferation, migration, and invasion in real-time by both H69 cholangiocytes and CC-LP-1 line of cholangiocarcinoma cells using the xCELLigence approach and Matrigel matrix revealed that exposure to ≥10 H. pylori bacilli per cell stimulated migration and invasion by the cholangiocytes. In addition, 10 bacilli of H. pylori stimulated contact-independent colony establishment in soft agar. These findings support the hypothesis that infection by H. pylori contributes to the malignant transformation of the biliary epithelium.

Histopathological findings and immunohistochemical expression of the stem cell markers CD44, ALDH1, Bmi-1, and Nanog in oral solitary fibrous tumors

The aim of this study was to evaluate the histomorphologic presentation and the expression of stem cell-related markers in a series of oral solitary fibrous tumors (SFTs). Histopathological variables and the expression of the standard stem cell markers CD34 and CD99, used for SFT diagnosis, as well as STAT6 were evaluated in 13 oral SFTs. The expression of the cancer stem cell markers CD44, ALDH1, Bmi-1, and Nanog and the tumor suppressor gene p16Ink4a were also investigated. The majority of oral SFTs were circumscribed and characterized by a proliferation of spindle cells arranged in a hyalinized stroma. Only 2 oral SFTs showed >4 mitoses/10 high-power fields. Hypercellularity as well as nuclear and cellular pleomorphism were classified as low and moderate in most of the oral SFTs. All oral SFTs were positive for CD34, STAT6, CD44, ALDH1, Bmi-1, and p16Ink4a. CD99 and Nanog expression was observed in 11 and 10 oral SFT cases, respectively. We suggest that STAT6 and ALDH1 have relevant diagnostic value. The expression of CD44, ALDH1, Bmi-1, and Nanog, which is observed in cancer stem cells, may confer advantages to oral SFT cells.

The prognostic value of combining CD133 and mismatch repair proteins in patients with colorectal cancer.

The prognostic value of cancer stem cells (CSCs) is a hot topic in colorectal carcinoma (CRC) research. CD133 has been identified as an important colorectal CSC marker, but its prognostic significance remains controversial. Recently, studies have reported a possible functional link between CSCs and DNA mismatch repair (MMR) system. However, the relationship between CRC stemness and MMR proteins remains little explored, and whether the predictive role of CD133 is affected by MMR proteins is still unknown. The aim of our study is to investigate the influence of MMR proteins on the predictive significance of CD133 in terms of CRC patient survival and to further analyze the correlation between MMR proteins and cancer stemness. In our study, we didn't observe the prognostic value of CD133 in CRC patients. However, we demonstrated that in patients with low expression of MSH6, MSH2, PMS2 and MLH1, especially MSH6, CD133 was an effective prognostic biomarker. Moreover, correlation analysis revealed a positive correlation between MSH6 and CD133 expression. In vitro studies supported our clinical data and showed that the expression of cancer-associated stemness markers CD133, BMI-1, OCT-4 and SOX-2 was significantly decreased in siRNA-MSH6/MLH1 CRC cells. Thus, our results demonstrated that MMR proteins might play an important role in modulating the stemness of CRC cells. MMR proteins might be a crucial determinant that can help to accurately identify tumour subclones that may benefit from using the CSC marker CD133 as a prognostic marker.

Upregulation of S100A10 in metastasized breast cancer stem cells.

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient‐derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44+ cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell–related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.

21P Double immunofluorescence (dIF) in analysing protein coexpression in tumours

Multiplex immunofluorescence is widely used nowadays for assessment of multiple mutually exclusive markers in one slide. It is especially helpful to distinguish various cell subpopulations based on expression of different markers in the tumor microenvironment. However, it is also a good way of studying coexpression of molecular markers of different cellular processes and signaling pathways in the same cell, including the same subcellular compartment, in tumor tissues obtained directly from patients. We used dIF to assess features of epithelial-mesenchymal transition (EMT), cancer stem cells (CSC) and their possible regulators (androgen (AR) and estrogen (ER) receptors, transforming growth factor beta receptor type II (TGFbRII)) in a training cohort of prostate cancer (PC) and endometrial cancer (EC) tissues. dIF staining of tissue sections showed that EMT markers E-cadherin and N-cadherin were coexpressed on the membranes of tumor cells, and cells expressing both markers were most likely representing those in EMT as a transitional metastable process. Opposingly, CSC markers CD44 and CD133 direct coexpression was not found at least in the studied cases, though both markers demonstrated membranous staining with slightly different patterns. ER alpha and beta subtypes were also coexpressed in the nuclei of a minor percent of cells in EC, mostly in stroma. AR and TGFbRII were simultaneously expressed in most PC tumor cells, the former in nucleus and the latter in cytoplasm, so no direct coexpression in the same compartment was seen. The described staining patterns obtained using dIF give additional information to the data that can be obtained with single marker staining with immunohistochemistry, so that interrelations between cellular proteins can be additionally analyzed as a prognostic or predictive biomarker. dIF is a good way of studying coexpression of markers in the same subcellular compartment. Thorough selection of fluorophores and filters enables assessment of staining of separate markers with further study of coexpression using image analysis software. Further quantitative assessment of studied markers in a larger sample cohort is needed to get data on possible links between EMT and CSC and their regulators in PC.

Autophagy induced by Helicobacter pylori infection is necessary for gastric cancer stem cell emergence

The main cause of gastric cancer is the infection by the bacterium Helicobacter pylori which induces a chronic inflammation and an epithelial-to-mesenchymal transition (EMT) leading to the emergence of cells with cancer stem cell (CSC) properties. However, the underlying mechanisms have not been fully characterized. Moreover, H. pylori modulates the host cell autophagic process, but a few studies have investigated the role of this process in tumoral transformation. The aim of this study was to determine whether H. pylori-induced autophagy has a role in CSC emergence. Autophagic flux in response to H. pylori infection was characterized in AGS cell line expressing the tandem-tagged mCherry-GFP-LC3 protein and using a ratiometric flow cytometry analysis. Then, AGS and MKN45 cell lines were treated with bafilomycin or chloroquine, two pharmaceutical well-known inhibitors of autophagy, and different EMT and CSC characteristics were analyzed. First, a co-expression of the gastric CSC marker CD44 and the autophagic marker LC3 in mice and human stomach tissues infected with H.pylori was observed. Then, we demonstrated in vitro that H.pylori was able to activate the autophagy process with a reduced autophagic flux. Finally, infected cells were treated with autophagy inhibitors, which reduced (i) appearance of mesenchymal phenotypes and migration ability related to EMT and (ii) CD44 expression as well as tumorsphere formation capacities reflecting CSC properties. In conclusion, all these data show that H.pylori-induced autophagy is implicated in gastric CSC emergence and could represent an interesting therapeutic target.

1973P Membranous vimentin (Vim) expression as a possible surrogate of cell surface vimentin in prostate cancer (PCa)

Vimentin (Vim) up-regulation is an important marker of epithelial-mesenchymal transition (EMT) in cancer cells. At the same time cell surface Vim has been shown to be a possible marker of cells with cancer stem cell (CSC) characteristics. Regarding this, membranous expression of Vim in tumor tissue may be considered as a surrogate marker of EMT and CSC in the tumor, and show a possible link between these two processes. In the present work we aimed to evaluate membranous Vim expression in prostate cancer (PCa). 36 cases of PCa (radical prostatectomy specimens, 1 representative block from each case) were analyzed. 4 μm thick sections were immunohistochemically stained with polyclonal rabbit anti-Vimentin antibodies (1:1000 dilution, ThermoFischer Scientific). For visualization of staining peroxidase method with diaminobenzidine as a chromogen was used. Staining was evaluated semiquantitatively with x20 magnification. Membranous Vim staining was present in 29 (80.6%) of cases, but was notably less frequent than cytoplasmic one. Staining was mostly patchy, but the entire cell membrane was stained. In most cases (n=20, 69.0% of cases with staining) only up to 5% of tumor cells were stained, in 5 (17.2%) – 5-20%, in 1 (3.4%) case -- 20-50%, more than 50% of tumor cells were stained also in 1 (3.4%) case. When staining intensity was considered, in most cases cells with moderate (present in 19 (65.5%) of cases) or strong (n=15, 51.7%) membranous Vim staining were present. Weighed staining index (WSI) was calculated for membranous Vim, the median of which being 6 (range 0-16) for all cases and 9 (3-16) for stained ones. When amount of stained cells was considered, median WSI was 6 (0-48) and 9 (3-16), respectively. No significant correlations were found with tumor stage and grade, as well as preoperative androgen deprivation therapy. Membranous Vim staining was present in most cases, but usually only occasional cells stained positively, that may correspond to data about low prevalence of cells with CSC phenotype in tumors. However, in some cases strong staining was present. Further assessment of membranous Vim correlation with CSC markers CD44 and CD133 expression in the same cohort is planned.

Abstract LB-200: Alpha-catulin as a marker of a specific population of invasive breast cancer cells

Abstract Breast cancer is the most commonly diagnosed cancer in women worldwide. The epithelial-to-mesenchymal transition (EMT) has been associated with breast cancer progression. However transient, reversible nature of EMT and the fact that only a small minority of carcinoma cells may undergo an EMT in primary tumors makes observing an EMT in vivo a great challenge. In our previous studies, we observed that the upregulation of alpha-catulin expression correlates with the transition of tumor cells from epithelial to mesenchymal morphology. Therefore we developed a reporter system, Catulin-GFP that allows us for the first time to isolate and characterize in vivo a small population of invasive triple negative breast cancer cells from the xenograft model and test their cancer initiating potential. It is a reporter driving GFP expression from catulin gene promoter. In vitro 3D invasion assay in matrigel confirmed specific localization of the GFP positive reporter cells in invading population. Whereas depletion of catulin results in the decreased invasive potential of the tumor cells. Furthermore localization of the GFP positive reporter cells overlaps with the expression of known EMT markers like Vimentin, N-cadherin and many more. In addition, flow cytometry analysis of those reporter cell lines showed that GFP signal partially overlaps with known breast cancer stem cell markers CD44+ and CD24- and that catulin depletion results in the change of CD44 expression pattern. Moreover spheres serial replating assay showed decreased stemness potential of catulin depleted spheres. MDA-231 - triple negative breast cancer cell line with catulin reporter was injected into a mammary fat pad of Nod.Scid mice. FACS analysis was performed on the tumors that formed after 8 weeks. GFP+/CD49f+ and GFP-/CD49f+ cells were collected and isolated RNA has been recently subjected to RNAseq analysis and pathway analysis has been performed. 1620 genes have been found to be downregulated and 1670 to be upregulated affecting many cancer invasion related pathways. Part of the tumors were cut and stained with antibodies for known EMT markers (N-cadherin, Vimentin, CD44, and others) to determine the localization of specific populations within the tumor. Moreover MDA-231 catulin knockdown cells with decreased invasive potential and control cells expressing TurboGFP were injected into a mammary fat pad of Nod.Scid mice. RNA from tumors formed after 8 weeks has been also subjected to RNAseq analysis in order to establish transcriptional profile of catulin deficient tumors. Our data show that the Catulin-GFP reporter system marks invasive cells that localize highly outermost in spheres as well as in whole tumors and allows us to perform transcriptional profiling of those cells for characterization of early invasion markers and potential therapeutic targets for breast cancer. Citation Format: Mateusz Gielata, Kamila Karpińska, Agnieszka Kobielak. Alpha-catulin as a marker of a specific population of invasive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-200.

Abstract 4151: STAT3 regulates chemo-resistance and cancer stemness in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a major global health problem and its treatment outcomes are limited by therapeutic resistance. Cancer stem cells are the roots of tumor growth, recurrence, metastasis and treatment resistance. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor well known to promote carcinogenesis in a number of tumors. The present study aims to investigate the role of STAT3 in HCC. Total STAT3 levels were examined by qRT-PCR in patients with primary HCC who underwent curative partial hepatectomy. Elevated STAT3 levels were significantly associated with poor recurrence-free survival and overall survival of HCC patients (log-rank test, P = 0.019 and 0.008, respectively). Immunohistochemistry (IHC) was performed to determine the activation status of STAT3 in HCC. Phosphorylated (p)-Y705 and p-S727 staining was detected in HCC tissues, albeit with varying patterns of expression. To investigate the efficacy of STAT3-targeted therapeutics in HCC, napabucasin, a cancer stemness inhibitor targeting STAT3-driven gene transcription, and the specific small interfering RNA (siRNA) were examined in HCC cell lines Hep3B, HepG2 and Huh7. Both napabucasin and siRNA prominently downregulated RNA and protein levels of total STAT3 and p-STAT3 of HCC cells. Napabucasin reduced viability, induced death, hindered migration, impaired colony and spheroid formation efficiency of HCC cells, as well as lowered expression levels of the cancer stem cell markers granulin-epithelin precursor (GEP) and CD133 in whole genome RNA sequencing analysis. Notably, napabucasin combined with 5-fluorouracil (5-FU) synergistically inhibited cell proliferation and induced apoptosis, suggesting its potential in sensitizing HCC cells to chemotherapeutic agents. In addition, napabucasin diminished the colony and spheroid formation abilities of chemo-resistant HCC cells (acquired resistance to 5-FU with over 10-fold increase) to a similar extent as their parental counterparts, indicating its potency in targeting chemo-resistant HCC cells via cancer stemness inhibition. Furthermore, napabucasin also suppressed HCC tumor growth in vivo, accompanied with reduced total STAT3 and p-STAT3 levels and proliferation (Ki-67). Overall, the present study highlighted the prognostic significance of STAT3 in HCC, and its functional role in the control of chemo-resistance and cancer stemness in HCC through targeted therapeutics. Citation Format: Carol Lee, Elaine Yee-Ling OR, Charing Ching-Ning Chong, Tan To Cheung, Iris Ming-Jing Xu, Linda Wing-Chi Ng, Marcus Hung-Lam Law, Peter Tin-Chung Li, Stephen Lam Chan, Anthony Wing-Hung Chan, Philip Chun Yeung, Kelvin Kwok-Chai Ng, Paul Bo-San Lai, Siu Tim Cheung. STAT3 regulates chemo-resistance and cancer stemness in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4151.

EMP2 Is a Novel Regulator of Stemness in Breast Cancer Cells.

Little is known about the role of epithelial membrane protein-2 (EMP2) in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment. In silico analysis of gene expression data demonstrated a correlation of EMP2 expression with known metastasis-related genes and markers of cancer stem cells (CSC) including aldehyde dehydrogenase (ALDH). In breast cancer cell lines, EMP2 overexpression increased and EMP2 knockdown decreased the proportion of stem-like cells as assessed by the expression of the CSC markers CD44+/CD24-, ALDH activity, or by tumor sphere formation. In vivo, upregulation of EMP2 promoted tumor growth, whereas knockdown reduced the ALDHhigh CSC population as well as retarded tumor growth. Mechanistically, EMP2 functionally regulated the response to hypoxia through the upregulation of HIF-1α, a transcription factor previously shown to regulate the self-renewal of ALDHhigh CSCs. Furthermore, in syngeneic mouse models and primary human tumor xenografts, mAbs directed against EMP2 effectively targeted CSCs, reducing the ALDH+ population and blocking their tumor-initiating capacity when implanted into secondary untreated mice. Collectively, our results show that EMP2 increases the proportion of tumor-initiating cells, providing a rationale for the continued development of EMP2-targeting agents.

Overexpression of RRM2 is related to poor prognosis in oral squamous cell carcinoma.

Ribonucleotide reductase M2 (RRM2) is a rate-limiting enzyme involved in DNA repair and synthesis. This study aimed to investigate the expression level, clinicopathological significance, and prognostic value of RRM2 in oral squamous cell carcinoma (OSCC). Human OSCC tissue microarrays were used to detect the expression of RRM2, cancer stem cell (CSC) markers CD44 and aldehyde dehydrogenase 1 (ALDH1), and the epithelial-mesenchymal transition (EMT) marker Slug. The correlation of RRM2 expression with clinicopathological parameters was evaluated. The effects of RRM2 on cell proliferation, migration, and apoptosis were investigated. Compared with normal and dysplastic tissues, the expression of RRM2 in human primary OSCC was significantly increased, and its overexpression was correlated with advanced pathological grade. The overall survival rate of patients with high RRM2 expression was lower than that of patients with low RRM2 expression. The overexpression of RRM2 was significantly associated with OSCC recurrence, and its overexpression was correlated with the CSC markers CD44 and ALDH1 and the EMT marker Slug. The expression of RRM2 promotes the proliferation and migration of human OSCC cells and inhibits apoptosis. Ribonucleotide reductase M2 may be a novel target in the diagnosis, prognosis, and therapy of OSCC.

Phytol Down-Regulates Expression of Some Cancer Stem Cell Markers and Decreases Side Population Proportion in Human Embryonic Carcinoma NCCIT Cells

Cancer stem cells (CSCs), a subgroup of cancer cells, have self-renewal capacity and differentiation potential and drive tumor growth. CSCs are highly-resistant to conventional chemo-radio therapy. Phytochemicals were shown to be able to eliminate CSCs. Phytol is a diterpene alcohol with demonstrated anticancer effects. The current study compared the effect of phytol with retinoic acid (RA) as a well-known inducers of CSC differentiation and cisplatin, a common chemotherapy drug, on CSC markers in human embryonic carcinoma NCCIT cells. NCCIT cells were exposed to 10 mM RA for 14 day to induce differentiation. Moreover, NCCIT cells were treated with IC50 dose of cisplatin (12 µM) and phytol (40 µM) for 7 day. Real-time PCR showed that phytol was more effective that RA and cisplatin in down-regulating the CSC markers OCT4, NANOG, SOX2, ALDH1, ABCB1, CD44 and CD133. Percentage of SP (13%) and ABCB1+ (0.34%) in NCCIT cells decreased to 7% and 0.1% respectively after treatment with phytol. A very small proportion of NCCIT cells were positive for CD44 (0.2%) and CD133 (0.48%) and this fraction did not change significantly after treatment with three agents. In conclusion, phytol has the greatest inhibitory effect on CSC population and markers than RA and cisplatin.

Expression of cancer stem cell markers CD24, EPHA1 and CD9 and their correlation with clinical outcome in epithelial ovarian tumours.

There has been variability between laboratories in the identification of cancer stem cells (CSCs) markers for epithelial ovarian cancer (EOC). We have evaluated three new surface markers for EOC to identify CSCs precisely. Three new putative CSCs specific surface markers CD9, CD24 and EPHA1 identified by a bioinformatics approach were evaluated in normal ovary, fallopian tube and ovarian tumours. The expression of CD9 alone was observed in normal ovarian surface epithelium and fallopian tube whereas CD24 and EPHA1 were not expressed (n= 5). CD24 was expressed in all tumours (N= 101) while CD9 and EPHA1 were expressed in 89 and 71 tumours, respectively. The statistical analysis showed significant correlation of the stage of the disease (p< 0.0001), type of surgery (p< 0.0001) and residual disease (p< 0.0001) with overall survival. Although expression of CD9, CD24 and EPHA1 was observed in the majority of tumours there was no significant correlation with outcome. In patients who underwent primary surgery, increased expression of CD24 significantly correlated with poor survival. The expression of CD24 was significantly reduced (p< 0.002) upon analysis of paired sections from patients prior to surgery and at interval debulking surgery (n= 16). These findings suggest that overexpression of these new markers may be useful in identifying and targeting ovarian CSCs and CD24 may be a putative CSCs marker in ovarian cancer.

Role of cancer stem cell markers ALDH1, BCL11B, BMI-1, and CD44 in the prognosis of advanced HNSCC

PurposeCancer stem cells (CSCs) are held accountable for the progress of head and neck squamous cell carcinoma (HNSCC). In the presented study, the authors evaluated the prognostic value of CSC markers in two particular HNSCC cohorts.MethodsThis two cohort study consisted of 85 patients with advanced stage HNSCC, treated with primary radio(chemo)therapy (pRCT), and 95 patients with HNSCC, treated with surgery and partially adjuvant radio(chemo)therapy. Overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) were assessed. Samples were assessed for the expression of different molecular stem cell markers (ALDH1, BCL11B, BMI‑1, and CD44).ResultsIn the pRCT cohort, none of the baseline patient and tumor features exhibited a statistically significant relation with survival in either the cohort or the human papillomavirus (HPV)-stratified subcohorts. High expression of BMI‑1 significantly decreased OS and DFS, while high expression of CD44 decreased all modes of survival. Multivariate analysis showed significant prognostic influence for all tested CSC markers, with high BMI‑1 and CD44 decreasing survival (BMI-1: OS, DFS, DSS; CD44: OS, DFS) and high ALDH1 and BCL11B showing a beneficial effect on survival (ALDH1: OS, DFS; BCL11B: OS, DSS). In the surgical cohort, classical prognosticators such as HPV status, R1 resection, and nodal status in HPV-negative HNSCC played a significant role, but the tested CSC markers showed no significant effect on prognosis.ConclusionAlthough validation in independent cohorts is still needed, testing for CSC markers in patients with advanced or late stage HNSCC might be beneficial, especially if many comorbidities exist or disease is irresectable. The findings might guide the development and earlier use of targeted therapies in the future.

Pentadecanoic Acid, an Odd-Chain Fatty Acid, Suppresses the Stemness of MCF-7/SC Human Breast Cancer Stem-Like Cells through JAK2/STAT3 Signaling.

Saturated fatty acids possess few health benefits compared to unsaturated fatty acids. However, increasing experimental evidence demonstrates the nutritionally beneficial role of odd-chain saturated fatty acids in human health. In this study, the anti-cancer effects of pentadecanoic acid were evaluated in human breast carcinoma MCF-7/stem-like cells (SC), a cell line with greater mobility, invasiveness, and cancer stem cell properties compared to the parental MCF-7 cells. Pentadecanoic acid exerted selective cytotoxic effects in MCF-7/SC compared to in the parental cells. Moreover, pentadecanoic acid reduced the stemness of MCF-7/SC and suppressed the migratory and invasive ability of MCF-7/SC as evidenced by the results of flow cytometry, a mammosphere formation assay, an aldehyde dehydrogenase activity assay, and Western blot experiments conducted to analyze the expression of cancer stem cell markers—CD44, β-catenin, MDR1, and MRP1—and epithelial–mesenchymal transition (EMT) markers—snail, slug, MMP9, and MMP2. In addition, pentadecanoic acid suppressed interleukin-6 (IL-6)-induced JAK2/STAT3 signaling, induced cell cycle arrest at the sub-G1 phase, and promoted caspase-dependent apoptosis in MCF-7/SC. These findings indicate that pentadecanoic acid can serve as a novel JAK2/STAT3 signaling inhibitor in breast cancer cells and suggest the beneficial effects of pentadecanoic acid-rich food intake during breast cancer treatments.

Abstract B047: Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity

Abstract Advanced glycation end-products (AGEs), are reactive metabolites produced endogenously as a consequence of glucose uptake during glycolysis. AGEs accumulate in tissues and organs as we grow older to promote multiple chronic disease phenotypes. AGE pathogenic effects are mediated through modification of protein function, genetic fidelity, stress responses and cellular signaling pathways. Critically, cancer disparity factors such as a sedentary lifestyle, obesity and an unhealthy diet are external influences that also contribute to the AGE accumulation pool in the body. This research group examined circulating and tumor AGE levels in clinical specimens of prostate cancer and identified a race specific, tumor-dependent pattern of accumulation. AGE levels were highest in aggressive tumors, especially those from men with African ancestry. Increased AGE levels correlated with an upregulation in the receptor for advanced glycation end products (RAGE) and activated NFkB. In a syngeneic subcutaneous prostate cancer mouse model, chronic consumption of AGE resulted in a 3-fold increase in tumor growth. Strikingly, dietary-AGE mediated increases in tumor growth were accompanied by a cytoplasmic accumulation of AR, elevation in MYC, RAGE, and AGE as well as increased cell proliferation. Cytoplasmic accumulation of AR is elevated in CRPC tissue and is an independent predictor of biochemical recurrence. Our data show that treatment of prostate cancer cells with AGEs induces a neuroendocrine differentiated phenotype by promoting a more mesenchymal morphology and increasing the expression of associated marker genes including ENO2, MYC and SYP and the prostate cancer stem cell marker CD44 as well as the downregulation of AR. The aberrant activation and recruitment of immune cells is a major pathogenic consequence of AGE accumulation, and a series of studies have highlighted the tumor-associated immune response as a critical pathway contributing to cancer disparity. Using patient-derived primary tumor cells, the investigators found that AGEs released into the extracellular matrix may recapitulate the tumor-associated immune response observed in ancestry-specific prostate tumor tissues. Further preliminary studies indicate that AGE treatment of prostate cancer cells can alter how cancer cells metabolize glucose to promote an aggressive phenotype. The investigators' studies support the concept that AGE metabolites represent a biologic consequence of the socioeconomic and environmental factors that promote cancer health disparity. As our understanding of tumor biology advances, it is becoming increasingly clear that these inter-related lifestyle factors have distinct molecular consequences on the biologic makeup of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as its earlier development or its progression to more aggressive disease. Citation Format: Pamela Woods, Bradley A. Krisanits, Dion Foster, Lourdes M. Nogueira, Laura Spruill, Marvella E. Ford, Michael B. Lilly, Victoria J. Findlay, David P. Turner. Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B047.

MicroRNA-93 targets WASF3 and functions as a metastasis suppressor in breast cancer.

Cancer cells with cancer stem cell (CSC) properties initiate both primary tumor formation and metastases at distant sites. Acquisition of CSC properties is highly associated with epigenetic alterations, including those mediated by microRNAs (miRNAs). We have previously established the breast cancer patient‐derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, we found that the expression levels of 3 miRNAs (miR‐25, miR‐93, and miR‐106b) in the miR‐106b‐25 cluster were much lower in the CD44+ human cancer cells metastasized to the liver than those at the primary site. Constitutive overexpression of miR‐93 suppressed invasive ability and 3D‐organoid formation capacity of breast cancer cells in vitro and significantly suppressed their metastatic ability to the liver in vivo. Wiskott‐Aldrich syndrome protein family member 3 (WASF3), a regulator of both cytoskeleton remodeling and CSC properties, was identified as a functional target of miR‐93: overexpression of miR‐93 reduced the protein level of WASF3 in breast cancer cells and WASF3 rescued the miR‐93‐mediated suppression of breast cancer cell invasion. These findings suggest that miR‐93 functions as a metastasis suppressor by suppressing both invasion ability and CSC properties in breast cancers.

Expression of cancer stem cells markers in urinary bladder urothelial carcinoma and its precursor lesions

Cancer stem cells (CSC) and their role in tumorigenesis of various solid tumors have been studied in past decades. Urothelial CSC were first identified 10 years ago and subsequent studies have been performed with the aim to identify reliable markers of CSC. So far, a few studies have investigated a relationship between CSC markers expression in urothelial carcinoma tissue and histopathological characteristics of the tumor. In our study, we evaluated an immunoexpression of the CSC markers CD24, CD44, CD66 and CD133 in tissue sections of urothelial carcinoma (all tumor grades and stages were included), urothelial carcinoma in situ and non-neoplastic urothelium, totally 218 specimens were enrolled. All studied molecules were expressed either in tumor tissue and non-neoplastic urothelium. Urothelial carcinomas of higher tumor grade and stage expressed molecules CD24 and CD133 significantly more frequently whereas molecules CD44 and CD66 did not show significant association with tumor histopathological features. Our results showed that studied molecules are not suitable for direct detection of CSC in urothelial carcinoma tissue sections, but an expression of molecules CD24 and CD133 is significantly related to urothelial carcinoma grade and stage, which are both important prognostic indicators and therefore an expression of these markers might have a potential prognostic value.

The Role of Cancer Associated Fibroblasts in Maintaining Cancer Stem Cells

Breast cancer is one of the leading causes of cancer related deaths in females. Current treatment options for breast cancer ultimately fail, resulting in cancer relapse. One explanation for recurrence after treatment is the presence of cancer stem cells (CSCs). CSCs have high proliferative potential and are able to initiate tumor growth. In order to target CSCs, it is important to understand how CSCs are maintained. Recent studies have shown that cancer‐associated fibroblasts (CAFs) aid in the maintenance of CSCs. CAFs are an abundant cell type in the tumor microenvironment and have been found to promote tumor growth and confer resistance to anti‐cancer drugs by supporting cancer stem cells. We hypothesized that CAFs promote and maintain the growth of cancer stem cells in a novel tumor model (MCaP). Mammary carcinoma cells (MCaP) were propagated in culture from a tumor that developed spontaneously in an aged mouse. Cancer cells, along with CAFs were expanded from the isolated tumor. Cancer cells were grown as a mixed culture with CAFs or alone after enrichment. A colony growth assay was used to assess the formation of CSCs, which grow as colonies. Cultures were fixed and stained with cytokeratin and colonies were counted manually. Colony size was quantified using ImageJ. Flow cytometry was performed to detect and quantify surface expression of CSC markers CD44 and CD24 in mixed cultures and isolated cancer cells. To investigate whether CAFs enable cancer cell resistance to chemotherapy, we measured the dose response to cyclophosphamide or paclitaxel. Immunofluorescence was performed on tissue sections of metastatic lymph node and primary tumor. Tissues were stained with cytokeratin, aldehyde dehydrogenase (ALDH), and alpha‐sma (SMA). From the colony growth assay we noticed that colony formation and the size of colonies were both increased when MCaP cells were cultured with CAFs. Flow cytometry showed that MCaP cancer cells grown with CAFs resulted in the appearance of a CD44 high/CD24low population. This CSC population was not seen in cancer cells grown alone. After treating cells with paclitaxel or cyclophosphamide, we saw no notable difference in the viability of cancer cells in the presence or absence of CAFs. Immunofluorescence staining showed ALDH was expressed in SMA‐positive CAFs, but not in cytokeratin‐positive cancer cells. Although we did not find that CAFs protected cancer cells from chemotherapy, CAFs appear to have a positive effect on the size and quantity of CSC colonies. In addition, we detected a CD44 high, CD24 low CSC population in mixed cultures. CAFs were found to express the CSC marker ALDH, with expression increasing as the size of the tumor increased. This finding suggests that CAFs may provide a niche for CSCs. In conclusion, these results suggest that CAFs play an important role in maintaining CSCs in the MCaP mouse model.Support or Funding InformationThe University of the Virgin Islands MARC grant (GM008422)and The Jones lab ACS Pilot grant (K22CA230315)

DAXX inhibits cancer stemness and epithelial\u2013mesenchymal transition in gastric cancer

BackgroundDAXX is a transcription repressor that has been implicated in several types of cancers, but its role in the development of gastric cancer remains unknown.MethodsWe analysed the expression of DAXX in 83 pairs of gastric cancer samples, including neoplastic and adjacent tissues, and correlated the expression levels with clinical stages. We also investigated the molecular mechanisms by which DAXX downregulation promotes cancer growth using both in vitro and in vivo models.ResultsDAXX was downregulated in advanced gastric cancer samples. The expression of DAXX inversely correlates with that of cancer stem cell markers CD44 and Oct4 in gastric cancer lines. DAXX overexpression in gastric cancer cells inhibited migration, invasion and epithelial– mesenchymal transition (EMT). The inhibition of EMT was achieved through the repression of SNAI3, a key inducer of EMT, by recruiting HDAC-1 into the nucleus. Using a xenograft mouse model, we demonstrated that the MKN45 cells formed smaller tumours when DAXX was overexpressed. Wild-type AGS cells were not able to form tumours in nude mice, but in contrast, formed visible tumours when DAXX was silenced in the cells.ConclusionWe for the first time demonstrated that DAXX functions as a tumour suppressor in gastric cancer by inhibiting stem cell growth and EMT.