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Background: Anisakis spp. are zoonotic nematodes causing mild to severe acute and chronic gastrointestinal infections. Chronic anisakiasis can lead to erosive mucosal ulcers, granulomas and inflammation, potential tumorigenic triggers. How Anisakis exerts its pathogenic potential through extracellular vesicles (EVs) and whether third-stage infective larvae may favor a tumorigenic microenvironment remain unclear. Methods: Here, we investigated the parasite's tumorigenic and immunomodulatory capabilities using comparative transcriptomics, qRT-PCR and protein analysis with multiplex ELISA on human intestinal organoids exposed to Anisakis EVs. Moreover, EVs were characterized in terms of shape, size and concentration using classic TEM, SEM and NTA analyses and advanced interferometric NTA. Results: Anisakis EVs showed classic shape features and a median average diameter of around 100 nm, according to NTA and iNTA. Moreover, a refractive index of 5–20% of non-water content suggested their effective biological cargo. After treatment of human intestinal organoids with Anisakis EVs, an overall parasitic strategy based on mitigation of the immune and inflammatory response was observed. Anisakis EVs impacted gene expression of main cytokines, cell cycle regulation and protein products. Seven key genes related to cell cycle regulation and apoptosis were differentially expressed in organoids exposed to EVs. In particular, the downregulation of EPHB2 and LEFTY1 and upregulation of NUPR1 genes known to be associated with colorectal cancer were observed, suggesting their involvement in tumorigenic microenvironment. A statistically significant reduction in specific mediators of inflammation and cell-cycle regulation from the polarized epithelium as IL-33R, CD40 and CEACAM1 from the apical chambers and IL-1B, GM-CSF, IL-15 and IL-23 from both chambers were observed. Conclusions: The results here obtained unravel intestinal epithelium response to Anisakis EVs, impacting host's anthelminthic strategies and revealing for the first time to our knowledge the host-parasite interactions in the niche environment of an emerging accidental zoonosis. Use of an innovative EV characterization approach may also be useful for study of other helminth EVs, since the knowledge in this field is very limited. [ABSTRACT FROM AUTHOR]

Breast cancer is the second most cancer worldwide in females. The primary factor responsible for tumor recurrence is the presence of breast cancer stem cells (BCSCs), which escape the chemo-radiotherapy. In this study, we have investigated the role of Secretory phospholipase-A2 Group 2A (sPLA 2 -IIA) that is overexpressed in BCSCs of MCF7 and MDA-MB-231 breast cancer cell lines. Further, overexpression of sPLA 2 -IIA revealed an increased EGFR/JNK/c-JUN/c-FOS signaling in BCSCs, while sPLA 2 -IIA knockdown significantly reduced the percentage of BCSCs and decreased signaling in both the cell lines. Importantly, sPLA 2 -IIA knockdown showed differentiation of BCSCs. Strikingly, PET imaging showed a decreased metastatic potential of BCSCs. Our study revealed a novel role of sPLA 2 -IIA in regulating BCSCs, which play a crucial role in regulating the differentiation and metastatic potential of BCSCs. [Display omitted] • SPLA2-IIA knockdown reduces the number of BCSCs. • Loss of sPLA2-IIA decreases the tumorigenic potential of BCSCs in NOD/SCID mice. • SPLA2-IIA knockdown enhances the differentiation potential of BCSCs. • SPLA2-IIA knockdown leads to a decrease in the metastatic potential of BCSCs. • SPLA2-IIA knockdown reduces the EGFR/JNK/c-JUN-c-FOS signaling in BCSCs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Through the isolation of canine amniotic membrane cells it was possible to verify that these cells do not present tumorigenic potential and present a positive immunophenotype fro CD105, PCNA and MHCI and negative for CD34 and MHC II. In addition, they are able to differentiate into adipogenic, osteogenic and chondrigenic lines. • Canine amniotic membrane cells have characteristics of mesenchymal stem cells. • Differentiation in the three embryonic lineages. • Biosafety verified for in vivo application. The amniotic membrane can be considered as one of the sources of isolation of these cells, since it is found in the fetal maternal interface and has low immunogenicity. Mesenchymal stromal/stem cells (MSCs) have not been identified in canine amniotic membrane (AMC). Therefore, our objective was to isolate, culture, characterize and differentiate cells derived from canine amniotic membrane (AMC) and to verify its immunological and tumorigenic potential. For this, 12 dogs fetuses of each gestational age 32, 43 and 55 days were used, and the isolation and culture of the AMC were performed. We observed that the cells presented fibroblastoid morphology and high confluence even after freezing. We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells, as well as being CD34- and CD105+. Regarding the immunological markers, we found that IL-1, IL-2, IL-6, IL-10 and MHC II were not expressed, whereas MHC I was expressed. After application of AMC cells in nude mice we can verify that there was no tumor formation. Based on this, we conclude that canine amniotic membrane is a good and accessible source for obtaining MSCs of low immunogenic and tumorigenic potential for veterinary therapeutic applications. [ABSTRACT FROM AUTHOR]

Cancer stem cells are a population of slow-cycling cells within the tumour bulk, with self-renewal capacity that attracts interest as a therapeutic target. In highly heterogeneous tumours, like pancreatic ductal adenocarcinoma (PDAC) however, the characterisation of cancer stem cells has led to controversial results due to the lack of consensus on specific markers. Here we investigated the characteristics of a population of pancreatic cancer tumorspheres derived from different human pancreatic cancer cell lines and a primary line from a genetically engineered KPC mouse model, using flow cytometry and western blotting to analyse surface and stemness markers. We analysed tumorspheres tumorigenic potential using anchorage-independent soft agar assay as well as their metabolic plasticity and chemoresistance. Pancreatic cancer tumorspheres display a heterogeneous pattern of surface and stemness markers, nevertheless they are characterised by an increased tumorigenic potential and higher chemoresistance. In addition, we have shown that pancreatic cancer tumorspheres have a unique metabolic profile with reduced metabolic potential. Together our results indicate that, despite the heterogeneity characterising pancreatic cancer tumorspheres, we can identify a functional vulnerability that represents a window for pharmacological intervention and development of novel therapeutic strategies. Image 1 [ABSTRACT FROM AUTHOR]

The wide array of biological properties attributed to the CCN family of proteins (Perbal in Lancet 363(9402):62-64, 2004) led me to reconsider the possible relationship and roles that these proteins may play as a team, instead of acting on their own as individual regulators in various signaling pathways. The dynamic model which I present in this review stems from the contribution of the biological properties that we established for CCN3, one of the three founding members of the CCN family, which was identified by our group as the first CCN protein showing growth inhibitory properties (1992), expressed mainly in quiescent cells (1996), and showing anti-tumor activities in several cellular models both ex vivo and in vivo. At the present time CCN3 is the only member of the family that has been reported to negatively act on the progression of the cell cycle. The unique dual localisation of CCN3 in the nucleus and outside cells, either at the membrane or in the extracellular matrix, that I first established in 1999, and that now appears to be shared by several other CCN proteins, is a unique essential feature which can no longer be ignored. Based on the structural and functional properties of CCN3, shared by most of the CCN family members, I propose an « all in one » concept in which CCN proteins are team members with specific functions that are aimed at the same goal. This model accounts both for the functional specificity of the various CCN proteins, their sequential and opposite or complementary effects in various biological context, and for the biological consequences of their physical interaction and biological cross-regulation. [ABSTRACT FROM AUTHOR]

Ataxia-telangiectasia mutated (ATM) protein kinase is a major guardian of genomic stability, and its well-established function in cancer is tumor suppression. Here, we report an oncogenic role of ATM. Using two isogenic sets of human colon cancer cell lines that differed only in their ATM status, we demonstrated that ATM deficiency significantly inhibits cancer cell proliferation, migration, and invasion. The tumor-suppressive function of ATM depletion is not modulated by the compensatory activation of ATR, but it is associated with B56γ2-mediated Chk1/p53/CD44 signaling pathways. Under normal growth conditions, the depletion of ATM prevents B56γ2 ubiquitination and degradation, which activates PP2A-mediated Chk1/p53/p21 signaling pathways, leading to senescence and cell cycle arrest. CD44 was validated as a novel ATM target based on its ability to rescue cell migration and invasion defects in ATM-depleted cells. The activation of p53 induced by ATM depletion suppresses CD44 transcription, thus resulting in epithelial-mesenchymal transition (EMT) and cell migration suppression. Our study suggests that ATM has tumorigenic potential in post-formed colon neoplasia, and it supports ATM as an appealing target for improving cancer therapy. [ABSTRACT FROM AUTHOR]

Increasing evidence has indicated that the splicing factor hnRNPA2B1 plays a direct role in cancer development, progression, gene expression, and signal transduction. Previous studies have shown that knocking down hnRNPA2B1 in breast cancer cells induces apoptosis, but the mechanism and other functions of hnRNPA2B1 in breast cancer are unknown. The goal of this study was to investigate the biological function, clinical significance, and mechanism of hnRNPA2B1 in breast cancer. The expression of hnRNPA2B1 in 92 breast cancer and adjacent normal tissue pairs was analyzed by immunohistochemical staining. Stable clones exhibiting knockdown of hnRNPA2B1 via small hairpin RNA expression were generated using RNA interference technology in breast cancer cell lines. The effects of hnRNPA2B1 on cell proliferation were examined by MTT and EdU assay, and cellular apoptosis and the cell cycle were examined by flow cytometry. A nude mouse xenograft model was established to elucidate the function of hnRNPA2B1 in tumorigenesis in vivo. The role of hnRNPA2B1 in signaling pathways was investigated in vitro. Our data revealed that hnRNPA2B1 was overexpressed in breast cancer tissue specimens and cell lines. Knockdown of hnRNPA2B1 reduced breast cancer cell proliferation, induced apoptosis, and prolonged the S phase of the cell cycle in vitro. In addition, hnRNPA2B1 knockdown suppressed subcutaneous tumorigenicity in vivo. On a molecular level, hnRNPA2B1 knockdown decreased signal transducer and activator of transcription 3 and extracellular-signal-regulated kinase 1/2 phosphorylation. We concluded that hnRNPA2B1 promotes the tumorigenic potential of breast cancer cells, MCF-7 and MDA-MB-231, through the extracellular-signal-regulated kinase 1/2 or signal transducer and activator of transcription 3 pathway, which may serve as a target for future therapies. [ABSTRACT FROM AUTHOR]

The key molecular events that contribute to tumorigenesis are incompletely understood. The aim of the present study was to characterize and compare the biological phenotypes of three human telomerase reverse transcriptase (hTERT) and/or human papillomavirus 16 E6 and E7-immortalized esophageal epithelial cell lines, NE2-hTERT (NE2), NE3-E6E7-hTERT (NE3) and NEcA6-E6E7-hTERT (NEcA6). The present study used soft-agar colony formation assays, tumorigenicity assays in nude mice, and cell proliferation, adhesion and migration assays to identify the biological characteristics of NE2, NE3 and NEcA6 cells. NE2 and NE3 cells exhibited characteristics of benign cells, such as the inability to grow in soft agar or form tumors in nude mice. By contrast, NEcA6 cells had undergone transformation, as demonstrated by the ability to grow in soft agar and form tumors in nude mice. In addition, NEcA6 cells exhibited increased migration and adhesion capabilities when compared with NE2 and NE3 cells. In order to identify mechanism(s) that may contribute to the altered biological phenotypes exhibited by these cells, the expression of three proteins involved in modulating cell migration [fascin, ezrin/radixin/moesin family proteins and phosphorylated-focal adhesion kinase (Tyr 397)], as well as the expression status and subcellular localization of three key focal adhesions components (paxillin, talin and kindlin-2) were examined. Paxillin, talin and kindlin-2 were localized to adhesive sites that connect F-actin with the extracellular matrix in transformed NEcA6 cells, but were distributed in a diffuse manner in NE2 and NE3 cells. Knockdown of kindlin-2 in NE3 and NEcA6 cells decreased cell adhesion, however, NEcA6 cells demonstrated a greater sensitivity to knockdown of kindlin-2. No significant differences were observed in the protein expression levels of fascin, exrin/radixin/moesin and p-FAK in the three cell lines. In conclusion, these results demonstrate that these three focal adhesion components, particularly kindlin-2, may contribute to the carcinogenesis of esophageal squamous cells. [ABSTRACT FROM AUTHOR]

Based on a publication of Tomasetti and Vogelstein in 2015, in which the risk of cancer development is postulated to be just one-third caused by genetic predisposition and environmental factors, it seemed worth focusing again on the value of test systems for screening chemicals for their carcinogenicity. This review aims to firstly summarize data on a host-mediated in vivo/in vitro assay developed by our working group to screen the tumorigenic potential of chemicals. Subsequently, in this article the importance and advantages of host-mediated in vivo/in vitro assays in general have been compared with in vivo and in vitro tests. The applicability of the host-mediated in vivo/in vitro assay system within broad screening strategies is discussed. The main intention of this review is to stimulate developments of newer approaches in the field of carcinogenic testing., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Highlights: [•] Four distinct subpopulations with varying stemness potential exist in oral cancer. [•] Transition of one fraction to other highlighted phenotypic switch and plasticity. [•] This study proposes a central dogma for epithelial carcinogenesis in oral cancer. [ABSTRACT FROM AUTHOR]

Highlights: [•] PBA showed tumor initiating activities. [•] CBA demonstrated both tumor initiating as well as promoting activities. [•] Prior treatment of AsA to PBA and CBA resulted in delayed onset of tumorigenecity. [•] Prior treatment of AsA application showed an enhanced latency period in CBA induced tumorigenicity. [•] Topical application of CBA significantly altered the levels of xenobiotic and antioxidant enzymes. [ABSTRACT FROM AUTHOR]

The neurotrophin receptors TrkA (NGF receptor) and TrkC (NT-3 receptor) have been shown to be important in staging disease and predicting progression and drug response for various neoplasias such as neuroblastoma, medulloblastoma and prostate cancer. Less is known about the role of the p75 neurotrophin receptor in cancer, but it influences metastatic potential in glioblastoma. To determine the effect of each neurotrophin receptor or co-receptor expression in tumorigenesis, we examined PC12 pheochromocytomas. PC12 wild type (TrkA+, p75++) were compared to three PC12-derived cell lines expressing varying levels of TrkA or TrkC and/or p75. Growth rates, tumorigenic potential ex vivo and in vivo, and chemotherapeutic drug response profiles differed depending on the neurotrophin receptor phenotype. The ability of neurotrophins to rescue cells from doxorubicin or cisplatin induced cell death also varied depending on phenotype. Thus, unique neurotrophin receptor tumor profiles may determine tumor aggressiveness and chemoresistance. This work may help to develop tailored therapies for specific tumor phenotypes by combining traditional chemotherapy with neurotrophin receptor modulators. [ABSTRACT FROM AUTHOR]

Abstract: The International Agency for Research on Cancer (IARC) has classified a number of the chemical constituents reported in cigarette mainstream smoke (MS) as carcinogens. In the international literature, 81 IARC classified carcinogens have been reported historically in MS. Cigarette smoke is a complex aerosol of minute liquid droplets (termed the particulate phase) suspended within a mixture of gases (CO2, CO, NO x , etc.) and semi-volatile compounds. The gases and semi-volatiles are termed the vapor phase. Due to early difficulties in inducing carcinomas in laboratory animals following inhalation exposure to MS, the mouse dermal promotion assay became the standard method of comparing the tumorigenic potential of cigarette smoke condensates (the particulate phase of MS nearly devoid of MS gases and having a significant reduction of the semi-volatile components of the vapor phase). Of the 81 IARC carcinogens reported in MS, 48 are found exclusively in the particulate phase, 29 in the vapor phase only, and four IARC carcinogens in both phases. A general comparison of the quantity and potency of the individual carcinogenic constituents of the MS vapor and particulate phases illustrates that the potential carcinogenic contribution from the vapor phase might be significant. Therefore, the mouse dermal promotion assay may not be a sensitive comparator of the tumorigenic potential of different MSs displaying a diversity of vapor phase components. However, when used in a weight-of-the-evidence approach that includes smoke chemistry, in vitro studies using whole smoke and human exposure studies evaluating both vapor and particulate phase smoke constituents, the mouse dermal promotion assay remains an important risk assessment tool as the only test that reproducibly measures the tumorigenic potential of cigarette smoke condensate. [Copyright &y& Elsevier]

Abstract: Aflatoxin B1 (AFB1) has been classified as a category I human carcinogen, which is responsible for a high incidence of hepatocellular carcinoma. Since exposure to AFB1 can occur through skin contact in addition to ingestion and inhalation, the carcinogenic potential of topically applied AFB1 on mouse skin was investigated. Our results show that single topical application of AFB1 (80nmol) as a tumor initiator, followed by twice weekly application of 12-tetradecanoyl phorbol myristate acetate (TPA, 4nmol), resulted in tumor formation after 13 weeks. However, no tumorigenic potential was observed when AFB1 (16nmol) was used either as a complete carcinogen or as a tumor promoter (4nmol). Histological analysis of skin showed squamous cell carcinoma in the AFB1/TPA treated group. The application of AFB1 as a complete carcinogen, an initiator or a promoter after 24 weeks demonstrated widespread degenerative and necrotic changes in hepatic tissue as well, suggesting liver to be the target organ following percutaneous absorption. Additionally, twice weekly topical application of AFB1 caused significant induction of cutaneous CYP IA monoxygenases without any effect on hepatic levels while glutathione-S-transferase activity was induced more in the liver than skin. The topical application of AFB1 also resulted in increased hepatic and cutaneous lipid peroxidation with concomitant depletion of glutathione content. It is likely that due to higher induction of hepatic GST activity, products of lipid peroxidation may be detoxified and therefore unable to cause DNA damage making mice resistant to hepatic tumor formation. The overall results indicate a tumor initiating potential of AFB1 in mice and suggest that continued dermal exposure of AFB1, even at low doses, might lead to degenerative changes in hepatocytes. [Copyright &y& Elsevier]

The molecularly cloned myeloblastosis-associated virus type-1(N) (MAV-1(N)) strain induces specifically nephroblastomas in chicken. MAV-induced nephroblastoma constitutes a unique animal model of the human Wilms'' tumor. We have previously shown that the MAV-1(N) long terminal repeats (LTR) were necessary and sufficient for nephroblastoma induction. Since major determinants for oncogenesis have been mapped in the U3 region of several other retroviruses, we have analyzed the tumorigenic potential of five recombinant viruses partially deleted in their U3 region. The results obtained indicated that deletions of the LTRs resulted in a modification of the pathogenic spectrum of MAV-1(N) and a decreased efficiency for nephroblastoma induction. [Copyright &y& Elsevier]

To examine the relationship between altered spontaneous mutation rates and malignant characteristics of cells, two hydroxyurea-resistant Chinese hamster ovary cell lines, with alterations in ribonucleotide reductase, were examined for their rates of spontaneous mutation to 6-thioguanine and ouabain resistance, tumor growth rates and their ability to form experimental lung metastases. The most resistant cell line, H-R2T, showed no changes in the rate of spontaneous mutation to 6-thioguanine or ouabain resistance compared to the parental wild-type cell line; however, the mutant line formed lung metastases in experimental metastasis assays with BALB/c nu/nu mice, and exhibited metastatic abilities significantly different from the wild-type population. Furthermore, the H-R2T population did not show imbalances in any of the deoxyribonucleoside triphosphate pool sizes, which are frequently observed in cells altered in ribonucleotide reductase activity. The second hydroxyurea-resistant line, HN-AT, had gross alterations in dCTP and dGTP pools and although the rate of spontaneous mutation to 6-thioguanione resistance was unaltered, it showed a moderate decrease in the rate of spontaneous mutation to ouabain resistance when compared to the parental wild-type population. Interestingly, the HN-AT cell line did not form any lung metastases in the experimental metastasis assay. Both mutant cell lines, H-R2T, and HN-AT, had increased tumor growth rates in C57 BALB/c 'beige' nude (nu/nu) mice as compared to the parental wildtype population. In total, the results obtained with the two mutant cell lines question the association of altered mutation rates with increased metastatic potential. Although several explanations are possible for the altered malignant properties exhibited by H-R2T and HN-AT cells, it is interesting to note that the results are consistent with earlier suggestions that changes in ribonucleotide reductase may accompany modifications in the malignant characteristics of cells. [ABSTRACT FROM AUTHOR]

Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44 - CD133 - , CD44 - CD133 + , and CD44 + CD133 + subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44 + CD133 + cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44 + CD133 + Caco-2 cells contained mixed populations of CD44 + CD133 + and non-CD44 + CD133 + Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44 + CD133 + subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44 + CD133 + cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/β-catenin pathway was over-activated in CD44 + CD133 + cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/β-catenin signaling inhibitors. Our findings suggest that the CD44 + CD133 + subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Wnt signaling is involved in the regulation of cancer stem cells (CSCs); however, the molecular mechanism involved is still obscure. SFRP1, a Wnt inhibitor, is downregulated in various human cancers; however, its role in tumor initiation and CSC regulation remains unexplored. Here, we used a skin carcinogenesis model, which showed early tumor initiation in Sfrp1 -/- (Sfrp1 knockout) mice and increased tumorigenic potential of Sfrp1 -/- CSCs. Expression profiling on Sfrp1 -/- CSCs showed upregulation of genes involved in epithelial to mesenchymal transition, stemness, proliferation, and metastasis. Further, SOX-2 and SFRP1 expression was validated in human skin cutaneous squamous cell carcinoma, head and neck squamous cell carcinoma, and breast cancer. The data showed downregulation of SFRP1 and upregulation of SOX-2, establishing their inverse correlation. Importantly, we broadly uncover an inverse correlation of SFRP1 and SOX-2 in epithelial cancers that may be used as a potential prognostic marker in the management of cancer., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity is related to an increased risk of developing prostate cancer with high malignancy stages or metastasis. Recent results demonstrated that LOX-1, a receptor associated with obesity and atherosclerosis, is overexpressed in advanced and metastatic prostate cancer. Furthermore, high levels of oxLDL, the main ligand for LOX-1, have been found in patients with advanced prostate cancer. However, the role of LOX-1 in prostate cancer has not been unraveled completely yet. Here, we show that LOX-1 is overexpressed in prostate cancer cells and its activation by oxLDL promotes an epithelial to mesenchymal transition, through of lowered expression of epithelial markers (E-cadherin and plakoglobin) and an increased expression of mesenchymal markers (vimentin, N-cadherin, snail, slug, MMP-2 and MMP-9). Consequently, LOX-1 activation by oxLDL promotes actin cytoskeleton restructuration and MMP-2 and MMP-9 activity inducing prostate cancer cell invasion and migration. Additionally, LOX-1 increased the tumorigenic potential of prostate cancer cells and its expression was necessary for tumor growth in nude mice. In conclusion, our results suggest that oxLDL/LOX-1 could be ones of mechanisms that explain why obese patients with prostate cancer have an accelerated tumor progression and a greater probability of developing metastasis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine 73 (head-domain) and Serine 431 (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1 T14,Y15 , EIF4EBP1 T46,T50 , EIF4B S422 , AKT1S1 T246,S247 , CTTN1 T401,S405,Y421 , and CAP1 S307/309 in K8-S73A/D mutant and CTTN1 T401,S405,Y421 , BUB1B S1043 , and CARHSP1 S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYC S176 , ZYX S344 , and PNN S692 could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Increasing evidence has indicated that the splicing factor hnRNPA2B1 plays a direct role in cancer development, progression, gene expression, and signal transduction. Previous studies have shown that knocking down hnRNPA2B1 in breast cancer cells induces apoptosis, but the mechanism and other functions of hnRNPA2B1 in breast cancer are unknown. The goal of this study was to investigate the biological function, clinical significance, and mechanism of hnRNPA2B1 in breast cancer. The expression of hnRNPA2B1 in 92 breast cancer and adjacent normal tissue pairs was analyzed by immunohistochemical staining. Stable clones exhibiting knockdown of hnRNPA2B1 via small hairpin RNA expression were generated using RNA interference technology in breast cancer cell lines. The effects of hnRNPA2B1 on cell proliferation were examined by MTT and EdU assay, and cellular apoptosis and the cell cycle were examined by flow cytometry. A nude mouse xenograft model was established to elucidate the function of hnRNPA2B1 in tumorigenesis in vivo. The role of hnRNPA2B1 in signaling pathways was investigated in vitro. Our data revealed that hnRNPA2B1 was overexpressed in breast cancer tissue specimens and cell lines. Knockdown of hnRNPA2B1 reduced breast cancer cell proliferation, induced apoptosis, and prolonged the S phase of the cell cycle in vitro. In addition, hnRNPA2B1 knockdown suppressed subcutaneous tumorigenicity in vivo. On a molecular level, hnRNPA2B1 knockdown decreased signal transducer and activator of transcription 3 and extracellular-signal-regulated kinase 1/2 phosphorylation. We concluded that hnRNPA2B1 promotes the tumorigenic potential of breast cancer cells, MCF-7 and MDA-MB-231, through the extracellular-signal-regulated kinase 1/2 or signal transducer and activator of transcription 3 pathway, which may serve as a target for future therapies.

Cyclooxgenase-2 (COX-2) knock-out mouse experiments showed that COX-2 was necessary for in vivo allergic inflammation, such as passive cutaneous anaphylaxis, passive systemic anaphylaxis, and triphasic cutaneous allergic reaction. TargetScan analysis predicted COX-2 as a target of miR-26a and miR-26b. miR-26a/-26b decreased luciferase activity associated with COX-2-3'-UTR. miR-26a/-26b exerted negative effects on the features of in vitro and in vivo allergic inflammation by targeting COX-2. ChIP assays showed the binding of HDAC3 and SNAIL, but not COX-2, to the promoter sequences of miR-26a and miR-26b. Cytokine array analysis showed that the induction of chemokines, such as MIP-2, in the mouse passive systemic anaphylaxis model occurred in a COX-2-dependent manner. ChIP assays showed the binding of HDAC3 and COX-2 to the promoter sequences of MIP-2. In vitro and in vivo allergic inflammation was accompanied by the increased expression of MIP-2. miR-26a/-26b negatively regulated the expression of MIP-2. Allergic inflammation enhanced the tumorigenic and metastatic potential of cancer cells and induced positive feedback involving cancer cells and stromal cells, such as mast cells, macrophages, and endothelial cells. miR-26a mimic and miR-26b mimic negatively regulated the positive feedback between cancer cells and stromal cells and the positive feedback among stromal cells. miR-26a/-26b negatively regulated the enhanced tumorigenic potential by allergic inflammation. COX-2 was necessary for the enhanced metastatic potential of cancer cells by allergic inflammation. Taken together, our results indicate that the miR26a/-26b-COX-2-MIP-2 loop regulates allergic inflammation and the feedback relationship between allergic inflammation and the enhanced tumorigenic and metastatic potential., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cancer/testis antigen cancer-associated gene (CAGE) is known to be involved in various cellular processes, such as proliferation, cell motility, and anti-cancer drug resistance. However, the mechanism of the expression regulation of CAGE remains unknown. Target scan analysis predicted the binding of microRNA-200b (miR-200b) to CAGE promoter sequences. The expression of CAGE showed an inverse relationship with miR-200b in various cancer cell lines. miR-200b was shown to bind to the 3'-UTR of CAGE and to regulate the expression of CAGE at the transcriptional level. miR-200b also enhanced the sensitivities to microtubule-targeting drugs in vitro. miR-200b and CAGE showed opposite regulations on invasion potential and responses to microtubule-targeting drugs. Xenograft experiments showed that miR-200b had negative effects on the tumorigenic and metastatic potential of cancer cells. The effect of miR-200b on metastatic potential involved the expression regulation of CAGE by miR-200b. miR-200b decreased the tumorigenic potential of a cancer cell line resistant to microtubule-targeting drugs in a manner associated with the down-regulation of CAGE. ChIP assays showed the direct regulation of miR-200b by CAGE. CAGE enhanced the invasion potential of a cancer cell line stably expressing miR-200b. miR-200b exerted a negative regulation on tumor-induced angiogenesis. The down-regulation of CAGE led to the decreased expression of plasminogen activator inhibitor-1, a TGFβ-responsive protein involved in angiogenesis, and VEGF. CAGE mediated tumor-induced angiogenesis and was necessary for VEGF-promoted angiogenesis. Human recombinant CAGE protein displayed angiogenic potential. Thus, miR-200b and CAGE form a feedback regulatory loop and regulate the response to microtubule-targeting drugs, as well as the invasion, tumorigenic potential, and angiogenic potential.

Aim: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells., Methods: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line (LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, respectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA targeting ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analyses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) assay (in vitro) and tumour-growth assay (in vivo), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro)., Results: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest level of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expression was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cellular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shRNA-mediated ANXA2 silencing in vitro, and the tumour growth inhibition rate was 38.24% in vivo. The percentage of MHCC97-H cells in S phase dramatically decreased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells decreased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis., Conclusion: shRNA-mediated silencing of ANXA2 suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.