Putative Cancer Stem Cell Markers Research Articles

Putative Cancer Stem Cell Markers are Frequently Expressed by Melanoma Cells in Vitro and in Situ but are also Present in Benign Differentiated Cells.

Currently, there remains an incomplete view of cancer stem cells (CSCs) in solid tumours. We studied a panel of putative CSC surface markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) in 40 established melanoma cell lines and four early-passage melanoma strains by flow cytometry. We additionally examined 40 formalin-fixed paraffin-embedded melanoma tissues using immunofluorescence microscopy. This was compared with their expression in healthy skin, normal differentiated melanocytes and fibroblasts. Most of the putative CSC markers were expressed by both melanoma cell lines and tissues. When present, these proteins were expressed by the majority of cells in the population. However, the expression of these markers by cells in healthy skin sections, normal differentiated melanocytes, and fibroblasts revealed that differentiated non-malignant cells also expressed CSC markers indicating that they lack of specificity for CSCs. Culturing cell lines under conditions more characteristic of the tumour microenvironment upregulated CSC marker expressions in a proportion of cell lines, which correlated with improved cell growth and viability. The testing of melanoma cell lines (n = 40), early-passage cell strains (n = 4), and melanoma tissues (n = 40) showed that several putative CSC markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) are commonly present in a large proportion of melanoma cells in vitro and in situ. Further, we showed that these putative markers lack specificity for CSCs because they are also expressed in differentiated non-malignant cell types (melanocytes, fibroblasts, and skin), which could limit their use as therapeutic targets. These data are consistent with the emerging notion of CSC plasticity and phenotype switching within cancer cell populations.

Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma.

Cancer stem cells (CSCs) are a small sub-population of cells within a tumor mass proficient of tumor initiation and progression. Distinguishing features possessed by CSCs encompass self-renewal, regeneration and capacity to differentiate. These cells are attributed to the phenomenon of aggression, recurrence and metastasis in neoplasms. Due to their cancer initiating and contributing features, a proper understanding of these CSCs and its microenvironment would aid in better understanding of cancer and designing better targeted therapeutic strategies for improved clinical outcome, thus improving the prognosis. This article dispenses a narrative review of CSCs in the context of head and neck carcinoma under the sub headings of overview of cancer stem cells, methods of isolation of these cells, putative CSC markers of head and neck cancer, signaling pathways used by these cells and their therapeutic implications.

CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC)

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

Cell State Transitions and Phenotypic Heterogeneity in Luminal Breast Cancer Implicating MicroRNAs as Potential Regulators.

Luminal breast cancer subtypes respond poorly to endocrine and trastuzumab treatments due to cellular heterogeneity arising from the phenotype transitions, accounted for mainly by the loss of receptor expression. The origins of basal-like and human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer subtypes have been attributed to genetic and protein modifications in stem-like cells and luminal progenitor cell populations, respectively. The post-transcriptional regulation of protein expression is known to be influenced by microRNAs (miRNAs) that are deemed to be master regulators of several biological processes in breast tumorigenesis and progression. Our objective was to identify the fractions of luminal breast cancer cells that share stemness potentials and marker profiles and to elucidate the molecular regulatory mechanism that drives transitions between fractions, leading to receptor discordances. Established breast cancer cell lines of all prominent subtypes were screened for the expression of putative cancer stem cell (CSC) markers and drug transporter proteins using a side population (SP) assay. Flow-cytometry-sorted fractions of luminal cancer cells implanted in immunocompromised mice generated a pre-clinical estrogen receptor alpha (ERα+) animal model with multiple tumorigenic fractions displaying differential expression of drug transporters and hormone receptors. Despite an abundance of estrogen receptor 1 (ESR1) gene transcripts, few fractions transitioned to the triple-negative breast cancer (TNBC) phenotype with a visible loss of ER protein expression and a distinct microRNA expression profile that is reportedly enriched in breast CSCs. The translation of this study has the potential to provide novel therapeutic miRNA-based targets to counter the dreaded subtype transitions and the failure of antihormonal therapies in the luminal breast cancer subtype.

Possible role of ALDH1 and CD44 in lip carcinogenesis.

Lip squamous cell carcinoma (LSCC) accounts for 12% of all head and neck cancers. It is caused by chronic exposure to ultraviolet light solar radiation and related to previous actinic cheilitis (AC). This study aimed to investigate the immunostaining of the putative cancer stem cells (CSC) markers ALDH1 and CD44 in AC (n=30) and LSCC (n=20). ALDH1 positivity was found to be statistically higher in LSCC than in AC lesions (p=0.0045), whilst CD44 expression was statistically higher in AC than in LSCC lesions (p=0.0155). ALDH1+ cells in AC lesions were associated with specific clinical features: a younger age (<60 years old), the female gender, white skin, not smoking or consuming alcohol, and a fast evolution, and not associated with the chronic exposure to UV radiation (p<0.0001). CD44 positivity was associated with patients who were male, feoderm, smoked, consumed alcohol, underwent occupational exposure to UV-radiation, and demonstrated lesions with log-time evolution (p<0.0001). ALDH1 + cells were associated with mild dysplasia using a system from the World Health Organization (WHO), and with a low risk of malignant transformation, according to the binary system (p<0.0001). CD44+ cells were also associated with moderated dysplasia, according to the WHO system. In LSCC, ALDH1 + cells were positively associated with patients who were older (≥ 60 years old), smokers, and with those who consumed alcohol (p<0.0001). CD44 + cells in LSCC were associated with older (≥ 60 years old) patients as well, but also with female patients, white skin, non-smokers, and individuals who did not consume alcohol (p<0.0001), all of whom showed distinct patterns in pre- and malignant lesions of both markers. Additionally, in LSCC, both ALDH1 and CD44 staining were associated with smaller tumor sizes (T1/T2; p<0.0001). In summary, although both ALDH1 and CD44 were associated with the presence of dysplasia in AC lesions, the present findings suggest that ALDH1 and CD44 may be activated by different etiopathogenic pathways, predominantly in distinct steps of oral carcinogenesis. CD44 would thus be more significantly related to the potentially malignant lesion, while ALDH1 would be closely linked to malignancy.

CSIG-08. MASS CYTOMETRY IDENTIFIES LOCATION-SPECIFIC TUMOR POPULATION ASSOCIATED WITH CLINICAL PROGRESSION IN GLIOBLASTOMA

Abstract Recent advances in single cell expression technology have allowed us to appreciate the complexity in tumoral heterogeneity of solid tumors such as glioblastoma (GBM), which remains uniformly fatal despite aggressive therapies. Currently, there is no knowledge about cell identities and functional characteristics of the different tumoral populations. To this end, we created a 28-marker mass cytometry panel composed of signaling markers, including phospho-proteins, and lineage markers that have been associated with GBM or are considered putative cancer stem cell markers. We evaluated lines established from core and edge areas from the same patient and validated to be functionally different with RNA expression and in vivo experiments. We found clusters shared by cells from both regions as well as region-specific ones. In order to better characterize these, we used marker enrichment modeling (MEM) and generated machine-readable labels. These were compared to MEM labels generated after using the same signaling markers to cluster primary samples from newly diagnosed or recurrent patients. Similarly, MEM labels were obtained from patients in a clinical trial treated with capecitabine that had positive or negative responses. Clusters from edge cells were found in higher proportion in recurrent tumors and were correlated with negative responses to chemotherapy. Initial analyses have also been done correlating immune clusters with tumor clusters in the same samples. Edge clusters are characterized by a CD44+ SOX2+ Nestin+ population. Treatment of tumor cells with temozolomide and irradiation results in an increase of this population in vitro. We are currently conducting single cells analyses to characterize this population as well as in vivo studies to validate the tumor-immune interactions. Our studies exploit single cell cytometry to underscore pathways that are preferentially use by different populations and affect immune cells with the goal of establishing therapeutic efforts to extend patient survival.

Transcription factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression

BackgroundUbiquitin-specific protease 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in various human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown.MethodsA luciferase reporter driven by a promoter region of USP22 gene was selectively constructed to screen against a customized siRNA library targeting 89 selected transcription factors to identify potential transcription factors (TFs) that regulate USP22 expression in human non-small cell lung cancers (NSCLC). Association of identified TFs with USP22 and potential role of the TFs were validated and explored in NSCLC by biological assays and immunohistochemistry analysis.ResultsLuciferase reporter assays revealed that SP1 and activating transcription factor 3 (ATF3) inhibit USP22 transcription, while transcription factor AP-2 Alpha/Beta (TFAP2A/2B) and c-Myc promote USP22 transcription. Binding site-directed mutagenesis and chromosome immunoprecipitation (ChIP) assays validated AP2α and AP2β are novel TFs of USP22. Furthermore, overexpression of AP2A and AP2B significantly upregulates USP22 expression, and its target: Cyclin D1, concurrently enhances the proliferation, migration, and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. Moreover, AP2 protein level correlated with USP22 protein in human NSCLC tissues.ConclusionOur findings indicate AP2α and AP2β are important transcription factors driving USP22 gene expression to promote the progression of NSCLC, and further support USP22 as a potential biomarker and therapeutic target for lung cancer.1HTL99SXtPtQRdWEzUs9UTVideo

CD133, but Not CD44, May Serve as a Novel Biomarker for Differential Diagnosis Between Basal Cell Carcinoma and Trichoblastomas.

PurposeTo investigate the clinical value of CD133 and CD44 as putative cancer stem cell markers in distinguishing between basal cell carcinoma (BCC) and trichoblastomas (TB).Patients and MethodsTumor samples from 24 BCC and 23 TB patients were retrospectively retrieved for immunohistochemical staining of CD133 and CD44. The results were interpreted using a semiquantitative scoring system (H score). A receiver operating characteristic (ROC) curve was developed to identify an optimal cutoff value for differentiating between BCC and TB.ResultsExpression of CD133 was significantly higher in BCC patients than in TB patients (median H score: 30 [IQR: 12.5–56.3] vs 0 [IQR: 0–2], P < 0.001). However, there was no significant difference in CD44 expression between the two groups (median H score: 105 [IQR: 63.8–155.0] vs 60 [IQR: 30–120], P = 0.095). The ROC analysis of CD133 immunostaining yielded an area under the curve (AUC) of 0.881 (95% CI: 0.756–1.000) for differentiating between BCC and TB by using a H score of 7 as the cut-off value (98.5% sensitivity and 87.0% specificity). By contrast, immunostaining of CD44 showed a lower diagnostic value, with an AUC of 0.642 (95% CI: 0.476–0.808) at the optimal cut-off value of 85 (62.5% sensitivity and 73.9% specificity). The positive and negative predictive values were 88.5% and 95.2% for CD133 and 71.4% and 65.4% for CD44, respectively. Additionally, CD133 expression was significantly associated with mitotic activity in BCC patients (r = 0.549, P = 0.005).ConclusionOur study expanded upon previous studies of CD133 and CD44 expressions in skin tumors, suggesting that CD133, but not CD44, may serve as a novel biomarker for differential diagnosis of BCC, although future studies using a larger number of patients are needed to justify it further.

Significance of CD133 Expression in Invasive Ductal Carcinoma of the Breast

Background and Aim: Cancer stem cells (CSCs) are thought to be responsible for tumor initiation, progression, and resistance to chemotherapy and radiotherapy. CD133 is a trans-membrane glycoprotein which is considered as a putative CSCs marker. Emerging evidence suggests that CD133 may be a critical factor in tumor development, progression and metastasis. The aim of this study was to evaluate the expression of CD133 in mammary infiltrating ductal carcinoma (IDC), and to correlate its expression with some known clinicopathological parameters. Methods: Fifty patients with mammary IDC who underwent modified radical mastectomy were included in this study. From each specimen, two tissue sections were obtained; one was stained by hematoxylin and eosin (H&amp;E) stain to determine the histologic subtype, grade and indicators of local aggressiveness. The second tissue section was immunohistochemically stained by anti-human CD133 antibody. Results: The study revealed statistically significant associations between CD133 expression and poorly differentiated, advanced stage tumors with poor Nottingham Prognostic Index (NPI), triple negative phenotype, lymphovascular invasion (LVI) and lymph node metastasis (LNM). Conclusion: The current study revealed that CD133 is strongly associated with poor prognostic indices; it is positively correlated to poorly-differentiated tumors with high histologic grade and advanced stage&#x0D;

Significant co-expression of putative cancer stem cell markers, EpCAM and CD166, correlates with tumor stage and invasive behavior in colorectal cancer

BackgroundThe crucial oncogenic role of cancer stem cells (CSCs) in tumor maintenance, progression, drug resistance, and relapse has been clarified in different cancers, particularly in colorectal cancer (CRC). The current study was conducted to evaluate the co-expression pattern and clinical significance of epithelial cell adhesion molecules (EpCAM) and activated leukocyte cell adhesion (CD166 or ALCAM) in CRC patients.MethodsThis study was carried out on 458 paraffin-embedded CRC specimens by immunohistochemistry on tissue microarray (TMA) slides.ResultsElevated expression of EpCAM and CD166 was observed in 61.5% (246/427) and 40.5% (164/405) of CRC cases. Our analysis showed a significant positive association of EpCAM expression with tumor size (P = 0.02), tumor stage (P = 0.007), tumor differentiate (P = 0.005), vascular (P = 0.01), neural (P = 0.01), and lymph node (P = 0.001) invasion. There were no significant differences between CD166 expression and clinicopathological parameters. Moreover, the combined analysis demonstrated a reciprocal significant correlation between EpCAM and CD166 expression (P = 0.02). Interestingly, there was a significant positive correlation between EpCAM/CD166 phenotypes expression and tumor stage (P = 0.03), tumor differentiation (P = 0.05), neural, and lymph node invasion (P =0.01).ConclusionsThe significant correlation of EpCAM and CD166 expression and their association with tumor progression and aggressive behavior is the reason for the suggestion of these two CSC markers as promising targets to promote novel effective targeted-therapy strategies for cancer treatment in the present study.

Immunohistochemical expression of putative cancer stem cell markers aldehyde dehydrogenase 1, SOX2, CD44 and OCT4 in different grades of oral epithelial dysplasia.

The hypothesized existence of cancer stem cells (CSC) and its markers aldehyde dehydrogenase 1 (ALDH1), CD44, SOX2 and OCT4 in oral dysplastic tissues provides the potential for a more reliable assessment of malignant transformation of oral epithelial dysplasia (OED). Thus, the present study is intended to evaluate the immunohistochemical expression of four different CSC markers ALDH1, CD44, SOX2 and OCT4 in different grades of OED and to investigate the co-expression of these putative stem cell markers in OED. A total of 35 samples of varying grades of OED which included 7 mild, 11 moderate and 17 severe dysplasia samples and 10 samples of normal oral mucosa without dysplasia were used. Four sections each from all 45 samples were stained with ALDH1, CD44, SOX2 and OCT4, respectively, by immunohistochemistry. The acquired data were analyzed and evaluated using the Chi-square test and unpaired t-test and the P < 0.05 was taken significant. ALDH1 and SOX2 expression percentages showed statistically significant differences among study groups (P < 0.05). Statistical comparison of percentage expression of CD44 and OCT4 between OED and normal was nonsignificant (P > 0.05). Co-expression of all four markers was found in 15 cases of OED with none of the normal cases showing co-expression. The expression of CSC markers in OED and normal mucosa differs significantly with co-expression of all four markers located only in dysplastic tissues. Until now, no single protein marker has been able to unequivocally identify the CSCs. Thus, a panel of putative CSC markers will help in identifying the patients with high risk for malignant transformation in OED.

Upregulation of Ganglioside GD2 Synthase (GD2S), as a New Putative Cancer Stem Cell Marker in Breast Carcinomas.

Background: GD2 synthase (GD2S) is the key enzyme required for ganglioside GD2 synthesis. It is commonly expressed in normal tissues and various cancers. Ganglioside GD2 is identified as a breast cancer stem cells (BCSCs) marker that promotes tumorigenesis. As GD2S has been found to be a useful molecular marker in neuroblastoma and retinoblastoma tumors, we suggest that it can be considered as a suitable candidate for the detection of CSCs in breast cancer tissues. Methods: Expression of GD2S was examined in 65 breast tumors compared to adjacent normal tissues, applying quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The association between GD2S expression level and patients’ clinical characteristics was also assessed. Results: Our findings showed that GD2S mRNA expression was significantly higher in breast cancer tissues in comparison to normal adjacent tissue samples (4.92-fold change, p<0.001) in advanced grades (p<0.001) and stages (p<0.001). It was also shown that GD2S protein expression was significantly higher in breast cancer tissues in comparison to normal adjacent tissues (4.86-fold change, p=0.010) in advanced grades (p=0.010), stages (p=0.005) and larger tumor size (p=0.002). Conclusion: The current study showed that increased expression of GD2S in advanced breast cancer potentiates it as a promising tumor marker in these patients.

Human Papilloma Virus and Cancer Stem Cell markers in Oral Epithelial Dysplasia-An Immunohistochemical Study.

ObjectivesTo study the correlation between the putative cancer stem cell (CSC) markers aldehyde dehydrogenase 1 (ALDH1), cluster of differentiation 44 (CD44), sex-determining region Y-box 2 (SOX2), and octamer-binding protein 4 (OCT4) and human papilloma virus (HPV) infection using p16, the surrogate marker of HPV in oral epithelial dysplasia (OED) and normal mucosa.MethodsFive sections each from 40 histopathologically diagnosed cases of different grades of OED and 10 cases of normal oral mucosa without dysplasia were immunohistochemically stained with p16, ALDH1, CD44, SOX2, and OCT4, respectively.ResultsExpression of ALDH1 and SOX2 was significantly increased in OED cases, whereas CD44 and OCT4 expression was increased in normal mucosa. P16-positive OED cases showed upregulation of ALDH1 and OCT4 expression as compared to p16-negative cases, while CD44 and SOX2 expression was downregulated in p16-positive OED cases; however, the results were not statistically significant.ConclusionThe present study indicated a suggestive link between p16 and cancer stem cell marker expression in HPV-associated OED, and that p16 has a significant role in CSC progression in OED. This is the first study to evaluate the expression of putative CSC markers in HPV-associated OED. However, low study numbers are a potential limiting factor in this study.

Dual Knockdown of Musashi RNA-Binding Proteins MSI-1 and MSI-2 Attenuates Putative Cancer Stem Cell Characteristics and Therapy Resistance in Ovarian Cancer Cells.

In ovarian cancer, therapy resistance mechanisms complicate cancer cell eradication. Targeting Musashi RNA-binding proteins (MSI) may increase therapeutic efficacy. Database analyses were performed to identify gene expression associations between MSI proteins and key therapy resistance and cancer stem cell (CSC) genes. Then, ovarian cancer cells were subjected to siRNA-based dual knockdown of MSI-1 and MSI-2. CSC and cell cycle gene expression was investigated using quantitative polymerase chain reaction (qPCR), western blots, and flow cytometry. Metabolic activity and chemoresistance were assessed by MTT assay. Clonogenic assays were used to quantify cell survival post-irradiation. Database analyses demonstrated positive associations between MSI proteins and putative CSC markers NOTCH, MYC, and ALDH4A1 and negative associations with NOTCH inhibitor NUMB. MSI-2 expression was negatively associated with the apoptosis regulator p21. MSI-1 and MSI-2 were positively correlated, informing subsequent dual knockdown experiments. After MSI silencing, CSC genes were downregulated, while cell cycle progression was reduced. Metabolic activity was decreased in some cancer cells. Both chemo- and radioresistance were reduced after dual knockdown, suggesting therapeutic potential. Dual knockdown of MSI proteins is a promising venue to impede tumor growth and sensitize ovarian cancer cells to irradiation and chemotherapy.

Abstract 2435: Transcription Factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression

Abstract Ubiquitin-specific peptidase 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in various human cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in a number of human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown. We set out to characterize and identify potential transcription factors that regulate USP22 expression in human non-small cell lung cancers (NSCLC). A luciferase reporter driven by the promoter region from -5085 to +55 of USP22 gene was constructed to screen a customized siRNA library targeting about 100 selected transcription factors that are expected to bind to the USP22 promoter. Luciferase reporter assays have revealed that knockdown of SP1 and activating transcription factor 3 (ATF3) promote USP22 expression, while knockdown of transcription factor 2α, 2β (TFAP2α and TFAP2β), cMyc, and PPAR α suppress USP22 expression. The specific binding of TFAP2α and TFAP2β to the USP22 promoter was further validated by binding site-directed mutagenesis and chromosome immunoprecipitation (Chip) assays. Furthermore, overexpression of TFAP2α and TFAP2β significantly up-regulate USP22 expression and downstream molecules of USP22, concurrently enhance the proliferation, migration and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. In addition, TFAP2 was demonstrated to be associated with a poor prognosis of NSCLC patients. Moreover, TFAP2 protein expression correlated with USP22 protein expression in human NSCLC tissues. Taken together, the findings of this study indicate TFAP2 is an important transcription factor driving USP22 gene expression, and it promotes the progression of NSCLC partially through enhancing USP22 expression. Citation Format: Keqiang Zhang, Ting Sun, Wendong Li, Rajendra Pangeni, Dan Raz. Transcription Factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2435.

ALDH1 expression predicts progression of premalignant lesions to cancer in Type I endometrial carcinomas

In type 1 endometrial cancer, unopposed estrogen stimulation is thought to lead to endometrial hyperplasia which precedes malignant progression. Recent data from our group and others suggest that ALDH activity mediates stemness in endometrial cancer, but while aldehyde dehydrogenase 1 (ALDH1) has been suggested as a putative cancer stem cell marker in several cancer types, its clinical and prognostic value in endometrial cancer remains debated. The aim of this study was to investigate the clinical value of ALDH1 expression in endometrial hyperplasia and to determine its ability to predict progression to endometrial cancer. Interrogation of the TCGA database revealed upregulation of several isoforms in endometrial cancer, of which the ALDH1 isoforms collectively constituted the largest group. To translate its expression, a tissue microarray was previously constructed which contained a wide sampling of benign and malignant endometrial samples. The array contained a metachronous cohort of samples from individuals who either developed or did not develop endometrial cancer. Immunohistochemical staining was used to determine the intensity and frequency of ALDH1 expression. While benign proliferative and secretory endometrium showed very low levels of ALDH1, slightly higher expression was observed within the stratum basalis. In disease progression, cytoplasmic ALDH1 expression showed a step-wise increase between endometrial hyperplasia, atypical hyperplasia, and endometrial cancer. ALDH1 was also shown to be an early predictor of EC development, suggesting that it can serve as an independent prognostic indicator of patients with endometrial hyperplasia with or without atypia who would progress to cancer (p = 0.012).

Effects of Total Western Diet and Wheat Class and Fraction on Preneoplastic Colon Cancer Risk

Abstract Objectives Colon cancer (CC) is a leading cause of cancer-related deaths worldwide and is particularly prevalent among persons consuming a Western-style diet. Red wheat, compared to white wheat, may reduce CC risk, as measured by reductions in colonic preneoplastic lesions (aberrant crypt foci; ACF). Rodent studies typically use a purified diet (AIN-93G) as the background diet, but due to its optimal nutritional composition, it may mask some effects of chemopreventive bioactives. The Total Western Diet (TWD), matched to the 50th percentile of US diets using NHANES data, has greater translational integrity to humans. This study aims to elucidate effects of background diet (AIN-93G vs TWD), wheat class (red vs white), and wheat fraction (whole vs refined vs testa layer) when fed during the post-initiation period. Methods Male Wistar rats (n = 83) were injected with the colon-specific carcinogen 1,2-dimethylhydrazine (twice, one week apart) to induce ACF. Five days after final injection, diets containing either AIN-93G or TWD background and various fractions of red and white wheat were fed for 10 weeks. Results No statistically significant differences in ACF number were found due to background diet. However, a statistically significant decrease in ACF was found in rats fed the TWD + whole red wheat (RW) relative to the TWD and the AIN-93G + refined red wheat diets. The short-chain fatty acid (SCFA) butyrate may act as a histone deacetylase to prevent CC. TWD + RW significantly increased total SCFAs as well as total butyrate compared to all other groups. TWD had significantly decreased total SCFAs and total butyrate compared to AIN-93G. Preliminary immunohistochemical results show that neither beta-catenin (part of the Wnt signaling pathway frequently dysregulated in CC) nor doublecortin-like kinase 1 (DCLK1; a putative cancer stem cell marker) staining of ACF significantly differ between TWD and TWD + RW. Conclusions The butyrate amount in cecal contents did not correlate with the staining intensity within ACF for beta-catenin or DCLK1 biomarkers, which does not support a role for high total butyrate reducing the risk of CC. Red wheat, when fed as part of the TWD, may reduce CC risk. Funding Sources Healthy Foods, Healthy Lives Institute, University of Minnesota.

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells.

Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase-dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1OE -sEVs), promote the migration of parental (non-transfected) MKN1 cells (MKN1PAR ). Quantitative proteome analysis of MKN1OE -sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1PAR -sEVs. Moreover, using DCLK1-IN-1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase-dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1-kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.

SMAD4 Expression in Renal Cell Carcinomas Correlates With a Stem-Cell Phenotype and Poor Clinical Outcomes.

Renal cell carcinoma (RCC) is the most lethal neoplasm of common urologic cancers with poor prognoses. SMAD4 has a principal role in TGF-β (Transformis growth factorβ)-induced epithelial to mesenchymal transition (EMT) as a key factor in gaining cancer stem cell (CSC) features and tumor aggressiveness. This study aimed to evaluate the expression patterns and clinical significance of SMAD4 in RCC and the impact of its targeting on stem cell/mesenchymal cells and EMT characteristics in renal spheroid derived cells (SDCs) compared to parental cells (PCs) in RCC. The expression pattern and clinical significance of SMAD4 was evaluated in RCC. SDCs were enriched using a sphere culture system. Then SDCs and their PCs were compared with respect to their sphere and colony formation, expression of putative CSC markers, invasiveness as well as expression of genes, including stemness/mesenchymal, SMAD4 and TGFβ1genes. Finally, the effect of SMAD4 knockdown on SDCs was analyzed. We demonstrated that SMAD4 is positively correlated with decreased disease specific survival (DSS) in RCC patients and clear cell RCC (ccRCC) subtype and associates with poor DSS in patients with RCC, especially in ccRCC as the most metastatic RCC subtype. SDCs exhibited higher stem cell/mesenchymal properties. Inhibition of SMAD4 in PCs accelerated the dissociation of SDCs and decreased their clonogenicity, invasiveness, expression of mesenchymal markers and expression of SMAD4 and TGFβ1 genes compared to SDCs before transfection. We suggest that targeting SMAD4 may be useful against renal CSCs and may improve RCC prognosis.

Expressions of Putative Cancer Stem Cell Markers, CD44 and CD133, are Correlated with Pathological Tumour Stage in Gastric Adenocarcinoma.

To analyse partial and total gastrectomy specimen in items of immunohistochemical staining of the stem cell CD44 and CD133; and to determine their relationship to pathological stage, other clinicopathological prognostic parameters, and their predictive value. Derscriptive study. Department of Pathology, Bolu Abant Izzet Baysal University, Medical School, Turkey, from 2013 to 2018. Eighty-three cases, diagnosed with adenocarcinoma from stomach partial and total gastrectomy slides and blocks, were included in the study. Adenocarcinoma cases, that had received neoadjuvant treatment before gastrectomy, cases diagnosed with lymphoma and other malignancies and cases of resection performed for reasons other than tumours, were excluded. Formalin-fixed and paraffin wax-embedded gastric adenocarcinoma blocks were sliced. CD44 and CD133 were stained onto the slides in a Leica Bond Max staining device in compliance with immunohistochemical staining datasheets. Membranous staining for CD44 and cytoplasmic, membranous or luminal staining for CD133 were evaluated. Mann-Whitney U-test and Kruskal- Wallis test were used for group comparisons. For pairwise comparisons, post-hoc Dunn's tests were used. The results were assessed on the significance level of p <0.05. The analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Considering CD44 and CD133 expressions in terms of pathological stage, there was significantly more intense expression in the T3 and T4 cases (p=0.009, and p=0.002, respectively). Although CD44 is regarded to be associated with more prognostic parameters compared to CD133, both immunohistochemical stains were shown to be related to pathological stage. Thus, these may be guiding for determining the tumour depth. Key Words: Gastric adenocarcinoma, CD44, CD133, Stem cell marker, Prognosis.