Anchorage-independent Growth Capacity Research Articles

Abstract PO3-24-11: Impact of Circulating Extracellular Vesicles derived from Obese Women on Pro-Tumor Functions of Stromal Fibroblasts in Breast Cancer

Abstract The incidence of obesity has risen sharply worldwide, and growing evidence shows its impact on several malignancies, including breast cancer (BC). Excessive adiposity and high body mass index (BMI) significantly affect BC incidence, prognosis, and therapeutic response of patients. Indeed, obese women exhibit larger tumor size, limph node involvement, metastatic spread, and poorer survival outcomes. Preclinical and clinical studies indicated that the obese setting provides local and systemic modifications that may stimulate BC progression and influence tumor microenvironment, particularly composed of stromal fibroblasts (SF). The mechanisms behind this interplay are likely to be multifactorial and may involve important adipocyte-derived mediators, such as adipokines and growth factors. Aside from these soluble factors, extracellular vesicles (EVs), nanoscale lipid-bilayer enclosed vesicles released by a variety of cells, are emerging as powerful regulators of cell-to-cell communication able to promote BC initiation, growth, metastatic dissemination and drug resistance. However, the potential impact of obesity-derived EVs in affecting tumor-stimulatory features of SF has not been yet investigated. To this aim, circulating EVs were isolated from the serum of women categorized as normal weight (NW; 18 > BMI < 24.9 Kg/m2) and overweight/obese (OW/Ob; BMI > 25 Kg/m2). Immortalised human mammary control fibroblasts (CF), their cancer-associated counterparts (exp-CAFs), and human CAFs, isolated from biopsies of primary BC, were used as experimental models for stromal fibroblasts. Human MCF-7 and T47D BC cells were employed as experimental cell models for mammary carcinoma in co-culture experiments. Circulating EVs were successfully extracted from the serum of NW and OW/Ob women, and characterized by their shapes, size and protein markers. Transmission Electron Microscopy (TEM), and Nanoparticle Tracking Analysis (NTA) showed that EVs in the isolated fractions were oval or bowl-shaped with a size range between 50-200 nm. In accordance with the Minimal Information for Studies of Extracellular Vesicles (MISEV) 2018, enrichment of the EV markers TSG101, CD81, and CD63 were all detected in the EV isolated fractions. Interestingly, elevated levels of circulating EVs were detected in OW/Ob women compared to NW subjects. Treatment of CF with OW/Ob-EVs resulted in an increased cell motility, invasiveness, contractility along with an up-regulation of CF markers. BC cells cocultured with conditioned medium (CM) derived from OW/Ob-EV-treated CF exhibited higher anchorage-independent growth and migratory capacity than BC cells cocultured with CM derived from NW-EV-treated CF. Moreover, OW/Ob-EVs enhanced tumor-stimulatory activities of exp-CAFs and CAFs, further highlighting how obese-related EVs may have the potential to modulate fibroblast phenotype in BC. Finally, cytokine arrays revealed that treatment of CF with OW/Ob EVs was associated with an enhanced release of Fibroblast Growth Factor 19 and Cystatin C, factors that are closely related to BC growth and development. In conclusion, our data provide first evidence for the functional importance of EVs isolated from obese women in impacting tumor-stroma cross-talk and drive BC progression. Since obesity and its pathophysiological sequelae are on the rise, this new knowledge may help to identify specific biomarkers and innovative targets (i.e. FGF-19, and Cystatin C) that may allow a personalized management of patients affected by BC and obesity. Citation Format: Ines Barone, Piercarlo Del Console, Felice Maria Accattatis, Luca Gelsomino, Giuseppina Augimeri, Ennio Avolio, Sebastiano Andò, Daniela Bonofiglio, Cinzia Giordano, Stefania Catalano. Impact of Circulating Extracellular Vesicles derived from Obese Women on Pro-Tumor Functions of Stromal Fibroblasts in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-11.

Alcohol consumption modulates Candida albicans-induced oral carcinogenesis and progression

ObjectivesThis study aimed to determine the impact of low levels of alcohol consumption on the interaction of the oral cavity with Candida albicans, a species that is commonly found at higher levels in the oral cavities of regular alcohol consumers, patients with pre-malignant diseases, and patients with existing oral cancer (OC). MethodsThe gingival squamous cell carcinoma cell line, Ca9-22, was subjected to low-level ethanol exposure before co-culture with heat-inactivated C. albicans (HICA). We performed cell viability assays, measured reactive oxygen species, and used Western blot analysis for cell death markers to examine the effect of ethanol and HICA on cells. Scratch assays and anchorage-independent growth assays were used to determine cell behavioral changes. ResultsThe results showed that ethanol in combination with HICA exacerbated cell death and cell cycle disruption, delayed NF-κB signaling, increased TIMP-2 secretion, and subsequently decreased MMP-2 secretion when compared to exposure to HICA alone. Conversely, both ethanol and HICA independently increased proliferation of Ca9-22 cells in scratch assays, and in combination, increased their capacity for anchorage-independent growth. ConclusionLow levels of ethanol may provide protective effects against Candida-induced inflammatory oral carcinogenesis or OC progression.

Glyphosate mimics 17β-estradiol effects promoting estrogen receptor alpha activity in breast cancer cells

Glyphosate, the active ingredient in several broad-spectrum herbicide formulations, has been validated and widely used throughout the world. Recent reports have questioned its safety, showing that glyphosate may act as an endocrine disruptor by promoting estrogenic activity. However, the molecular mechanism involved in this phenomenon remains unclear. Therefore, here we aimed to elucidate the mechanism by which glyphosate induces estrogenic activity using estrogen-sensitive breast cancer cell line models. Our results show that glyphosate mimics the cell effects of 17β-estradiol (E2), promoting estrogen receptor α (ERα) phosphorylation, its degradation, and transcriptional activity at high concentrations. The molecular mechanism seems involved in the ERα ligand-binding domain (LBD). Molecular simulations suggest a plausible interaction between glyphosate and the LBD through a coordinated complex involving divalent cations such as Zn (II). In addition, glyphosate exposure alters the level of Cyclin-dependent kinase 7 that contribute to ERα phosphorylation. Finally, glyphosate increases cell proliferation rate and levels of cell cycle regulators, accompanied by an increase in anchorage-independent growth capacity. These findings suggest that glyphosate at high concentrations, induces estrogen-like effects through an ERα ligand binding site-dependent mechanism, leading to cellular responses resulting from a complex interplay of genomic and non-genomic events.

Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG—a human glioblastoma-derived cell lineage—reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/β-catenin signaling pathways, in addition to upregulation of genes related to epithelial–mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

A Novel Role for Brain and Acute Leukemia Cytoplasmic (BAALC) in Human Breast Cancer Metastasis.

Brain and Acute Leukemia, Cytoplasmic (BAALC) is a protein that controls leukemia cell proliferation, differentiation, and survival and is overexpressed in several cancer types. The gene is located in the chromosomal region 8q22.3, an area commonly amplified in breast cancer and associated with poor prognosis. However, the expression and potential role of BAALC in breast cancer has not widely been examined. This study investigates BAALC expression in human breast cancers with the aim of determining if it plays a role in the pathogenesis of the disease. BAALC protein expression was examined by immunohistochemistry in breast cancer, and matched lymph node and normal breast tissue samples. The effect of gene expression on overall survival (OS), disease-free and distant metastasis free survival (DMFS) was assessed in silico using the Kaplan-Meier Plotter (n=3,935), the TCGA invasive breast carcinoma (n=960) and GOBO (n=821) data sets. Functional effects of BAALC expression on breast cancer proliferation, migration and invasion were determined in vitro. We demonstrate herein that BAALC expression is progressively increased in primary and breast cancer metastases when compared to normal breast tissue. Increased BAALC mRNA is associated with a reduction in DMFS and disease-free survival, but not OS, in breast cancer patients, even when corrected for tumor grade. We show that overexpression of BAALC in MCF-7 breast cancer cells increases the proliferation, anchorage-independent growth, invasion, and migration capacity of these cells. Conversely, siRNA knockdown of BAALC expression in Hs578T breast cancer cells decreases proliferation, invasion and migration. We identify that this BAALC associated migration and invasion is mediated by focal adhesion kinase (FAK)-dependent signaling and is accompanied by an increase in matrix metalloproteinase (MMP)-9 but not MMP-2 activity in vitro. Our data demonstrate a novel function for BAALC in the control of breast cancer metastasis, offering a potential target for the generation of anti-cancer drugs to prevent breast cancer metastasis.

Ex vivo exposure to different types of graphene-based nanomaterials consistently alters human blood secretome

The biomedical applications of graphene-based nanomaterials (GBN) have significantly grown in the last years. Many of these applications suppose their intravenous exposure and, in this way, GBN could encounter blood cells triggering an immunological response of unknown effects. Consequently, understanding the relationships between GBN and the immune system response should be a prerequisite for its adequate use in biomedicine. In the present study, we have conducted a little explored ex vivo exposure method in order to study the complexity of the secretome given by the interactions between GBN and blood cells. Blood samples from different healthy donors were exposed to three different types of GBN widely used in the biomedical field. In this sense, graphene oxide (GO), graphene nanoplatelets (GNPs), graphene nanoribbons (GNRs) and a panel of 105 proteins representatives of the blood secretome were evaluated. The results show broad changes in both the cytokines number and the expression levels, with important changes in inflammatory response markers. Furthermore, the indirect soft-agar assay was used as a tool to unravel the global functional impact of the found secretome changes. Our results indicate that the GBN-induced altered secretome can modify the natural anchorage-independent growth capacity of HeLa cells, used as a model. As a conclusion, this study describes an innovative approach to study the potential harmful effects of GBN, providing relevant data to be considered in the biomedical context when GBN are planned to be used in patients.

Abstract 4690: ARD1-mediated NRF2 acetylation promotes human colon cancer cell proliferation

Abstract The transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2) is regarded as one of the main orchestrators of the cellular antioxidant and carcinogen-detoxifying response. NRF2 plays a pivotal role in protecting normal cells from carcinogenic insults, but it may also accelerate the proliferation and progression of cancer cells. Abnormally elevated expression or activation of NRF2 has been attributed to mutation, especially in the sequences involved in interaction with its negative regulator KEAP1. However, other mechanisms responsible for NRF2 overactivation/overexpression are less well understood. Arrest defective (ARD1), an N-terminal acetyltransferase that catalyzes N-terminal acetylation of target proteins, has lysine acetyltransferase activity and is involved in mediating various (patho)physiological processes, such as proliferation, apoptosis, autophagy, and differentiation. Aberrant overexpression of ARD1 is correlated with metastasis and poor prognosis in several types of cancer, suggesting that it may act as a tumor promoter. In the present study, we found that NRF2, acteyl-NRF2, and ARD1 were highly expressed in some human colon cancer cell lines (HCT-116, HCT-15, DLD1) and tissues obtained from colorectal cancer patients. Furthermore, overexpression of NRF2 and ARD1 was also verified by quantitative immunofluorescence analysis of colorectal tissue microarray. We noticed that silencing of NRF2 and ARD1 individually in human colon cancer cells (HCT-116) led to the decreases in viability, anchorage-independent growth, and migration capacity of HCT-116 cells. While there was no change in the protein level of ARD1 when NRF2 expression was knock down by siRNA, protein expression of Nrf2 and acteyl-NRF2 was markedly reduced by silencing of the ARD1 gene. This prompted us to explore the association between ARD1 and NRF2 in the human colon cancer cell proliferation and progression. Co-immunoprecipitation and immunocytochemical analyses showed that two proteins colocalized in the cytosol, and ARD1 directly bound to NRF2. Taken together, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer cells by acetylating and stabilizing this transcription factor. This study was supported by the Global Core Research Center (GCRC) Grant (No. 2011-003-0001) from the National Research Foundation (NRF) of Republic of Korea. Citation Format: Xizhu Fang, Yeon-Hwa Lee, Jie Zheng, Seong Hoon Kim, Jeong-Hoon Jang, Do-Hee Kim, Young-Joon Surh. ARD1-mediated NRF2 acetylation promotes human colon cancer cell proliferation [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 4690.

Abstract B042: Loss of nuclear alpha catenin contributes to chemoresistance and aggressive disease in African American triple-negative breast cancer patients

Abstract Triple-negative breast cancer (TNBC) is a particularly aggressive, difficult-to-treat subtype of the disease. A well-documented health disparity exists within TNBC: African American (AA) women are more likely to be diagnosed with and die from the disease. Our group reported homozygous deletions in the CTNNA1 gene, which encodes the protein alpha catenin, in AA TNBC. We have undertaken a basic and translational research study to understand the mechanistic role and clinical impact of alpha catenin loss in TNBC, particularly in AA patients. To validate our findings of alpha catenin loss in TNBC, we analyzed over 600 breast cancer patient samples by immunohistochemistry. We found that alpha catenin protein loss was inversely correlated with survival in TNBC. Moreover, loss was observed more frequently in tumors from AA patients. Interestingly, both nuclear and cytosolic alpha catenin adhered to this pattern. While its cytosolic role has been well studied, little is known about nuclear alpha catenin and its role in disease. To understand the nuclear function of alpha catenin, we developed several isogenic cell line pairs. Using CRISPR/Cas9-mediated gene editing, we generated CTNNA1 knockout (KO) BT-549 and MB-MDA-436 cell lines. We also reintroduced CTNNA1 into MDA-MB-468 cells—a line derived from an AA woman with an endogenous deletion in CTNNA1. Using these models, we found that alpha catenin KO cells demonstrated greater capacity for anchorage-independent growth. Loss of alpha catenin also resulted in decreased sensitivity to DNA-damaging chemotherapies including platinum salts (cisplatin, carboplatin), topoisomerase poisons (doxorubicin, etoposide), and the PARP inhibitor olaparib. This is clinically relevant as doxorubicin, carboplatin, and olaparib are approved for treatment of TNBC patients. To identify binding partners, we performed co-immunoprecipitation of alpha catenin from nuclear lysates, followed by mass spectrometry. We found that nuclear alpha catenin can interact directly with ATR, a kinase critical to the DNA damage response (DDR). ATR mediates both the repair of DNA lesions and the G2/M cell cycle checkpoint, which ensures that only cells with undamaged DNA may enter mitosis. We further found that alpha catenin KO cells were more sensitive to inhibitors of ATR, as well as to inhibitors of the G2/M checkpoint proteins Chk1 and Wee1. Tellingly, the KO cells were less sensitive to inhibitors of ATM or DNA-PK, two regulators of alternate DDR pathways. This suggests that nuclear alpha catenin likely plays a specific role in ATR-directed processes. In our study, we have identified nuclear alpha catenin as a tumor suppressor that affects TNBC's susceptibility to chemotherapy by playing a role in the DDR and the G2/M checkpoint. Our data suggest that loss of alpha catenin is more common in AA patients, and is associated with poor prognosis. Therefore, CTNNA1 status may be important in determining appropriate therapeutic strategies in a subset of patients. Citation Format: Rania Bassiouni, Victoria Dirgo, Krystine Mansfield, Ritin Sharma, Lee D. Gibbs, Patrick Pirrotte, Nasreen Vohra, Kevin Gardner, John D. Carpten. Loss of nuclear alpha catenin contributes to chemoresistance and aggressive disease in African American triple-negative breast cancer patients [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 B042.

Establishment and characterization of immortalized porcine neonatal hepatocytes without the use of viral components.

Porcine hepatocytes are a promising option for xenotransplantation in light of the critical shortage of orthotopic donor livers. Because primary hepatocytes have limited ability to proliferate in vitro, several immortalized hepatocyte lines have been established. However, these cells have typically been generated using a virus-dependent transfection methodology and express viral oncogenes that introduce potential risks in clinical applications. In our study, we established immortalized porcine neonatal hepatocytes by introduction of a plasmid-based hTERT gene expression system by electroporation, without the use of viral components. We detected stable expression of hTERT by RT-PCR and Western blot. The immortalized hepatocytes exhibit a high growth rate, but retain the normal morphology of freshly isolated primary hepatocytes. To date, these immortalized hepatocytes have been expanded for over 80 passages. In addition, no significant differences were detected in glycogen synthesis, secretion of serum albumin, or lipid accumulation between the primary hepatocytes and our immortalized hepatocytes. The cells also exhibit serum-dependent growth and have no capacity for anchorage-independent growth in vitro, demonstrating that they have not been transformed in vitro. Our immortalized porcine hepatocytes will be useful for elucidating the pathogenesis of liver disease and developing efficient treatments. Furthermore, these immortalized hepatocytes may provide a safer source of cells for application in xenotransplantation, compared with immortalized cells generated using viral components.

PIK3CA Cooperates with KRAS to Promote MYC Activity and Tumorigenesis via the Bromodomain Protein BRD9.

Tumor formation is generally linked to the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed or contain a single or double knock-in (DKI) of PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC target gene signature. This gene signature was mediated in part by the bromodomain-containing protein 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced MYC transcript levels. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR-Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and BRD9 overexpression induced the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncoprotein cooperation and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS-driven oncogenic transformation.

Eckol inhibits Reg3A-induced proliferation of human SW1990 pancreatic cancer cells.

Pancreatic cancer (PaC) is characterized by a highly inflammatory tumor microenvironment, and inflammatory mediators are implicated in the progression of this cancer. Regenerating gene protein (Reg) 3A is significantly upregulated during pancreatic inflammation, and has been demonstrated to serve an important role during PaC progression, based on its increased expression levels in PaC and potent cell proliferation-promoting activity. The aim of the present study was to investigate the effect of eckol, a phlorotannin compound with a variety of biological activities including anti-inflammatory, anti-tumor and cytoprotective effects, on Reg3A-induced proliferation of human SW1990 PaC cells. SW1990 cells were pre-treated with eckol for 48 h at concentrations of 5, 10 and 20 µg/ml. Subsequently, Reg3A protein was added to the culture media at a final concentration of 50 ng/ml in the presence or absence of eckol for 24 h. The cytotoxicity and proliferative capacity of the SW1990 cells was determined using an MTT and flow cytometry analysis. Cell colony formation was also used to determine the effect of eckol on the anchorage-independent growth and colony-forming capacity of Reg3A-treated PaC cells. The expression levels of cyclin D1, STAT3, JAK2, and NF-κB p65 were measured with reverse transcription-quantitative PCR and western blotting. Eckol reduced Reg3A-promoted cell survival, inhibited Reg3A-induced cell cycle progression and inhibited colony growth of SW1990 cells in soft agar in a concentration-dependent manner. Additionally, the Reg3A-mediated upregulation of expression of JAK2, STAT3, NF-κBp65 and cyclin D1 was reduced when treated with eckol. Reg3A is upregulated during pancreatic inflammation and exhibits a pro-growth function and may thus serve a critical role during inflammation-driven PaC malignancies. Eckol may be a potential protective agent against progression of PaC accompanied by pancreatic inflammation.

Effect of low-dose tungsten on human thyroid stem/precursor cells and their progeny.

Thyroid cancer incidence is increased in volcanic areas where environment pollution biocontaminates residents. Tungsten (W) is the most increased heavy metal in drinking water of Mount Etna volcanic area where it exceeds the normal range in the urine of 27% inhabitants. The possible connection between increased tungsten and thyroid cancer has never been studied. We investigated in vitro the effect tungsten on both human thyrocytes in primary culture, thyrospheres (aggregates of stem/precursor thyroid cells) and thyrocytes differentiated from tungsten-exposed thyrospheres. Chronic exposure to low-dose (nanomolar range, as in the urines of volcanic area residents) soluble tungsten had major biological effects on thyroid stem/precursor cells, promoting growth with a biphasic (hormetic) dose-response and reducing apoptosis. No such effects were observed in mature thyrocytes. In addition, tungsten-exposed thyrospheres had abnormal expression of genes commonly altered also in thyroid cancer and increased activation of the DNA-repair proteins H2AX and 53BP1. Moreover, exposure to tungsten decreased thyrosphere differentiation, as indicated by the reduced expression of thyroid-specific genes in derived thyrocytes that also showed preneoplastic changes such as increased anchorage-independent growth, clonogenic growth and migration capacity. The mechanism of action of tungsten on thyroid stem/precursor cells is unclear but involves membrane G-proteins and activation of the ERK signaling pathway. These data indicate that chronic exposure to slightly increased tungsten, harmless for mature thyrocytes, importantly affects the biology of stem/precursor thyroid cells and of their progeny, inducing characteristics of preneoplastic transformation.

Differential Prognostic Relevance of Promoter DNA Methylation of CDO1 and HOPX in Primary Breast Cancer.

We previously identified that promoter DNA methylation of cysteine dioxygenase type 1 (CDO1) and homeobox only protein homeobox (HOPX) were both cancer specific, and have a clinical potential as prognostic biomarkers in breast cancer (BC). The present study compared the differential prognostic relevance of methylation status of the CDO1 and HOPX genes in BC. Methylation levels (TaqMethVs) were quantified in 7 BC cell lines and 133 BC patients by TaqMan methylation-specific PCR and functional traits were explored for CDO1. TaqMethVs were associated between CDO1 and HOPX (r2=0.072, p=0.002). Multivariate Cox proportional hazards model could identify CDO1 hypermethylation as well as Ki-67 as independent prognostic factors related to disease-specific survival (p=0.016, p<0.001). Overexpression of CDO1 decreased the anchorage-independent growth capacity in BC cell lines. CDO1 is a definite tumor suppressor gene, while its prognostic relevance was more than expected in the context of its functional relevance.

Radiation-Induced Malignant Transformation of Preneoplastic and Normal Breast Primary Epithelial Cells

Radiation is used in multiple procedures as a therapeutic and diagnostic tool. However, ionizing radiation can induce mutations in the DNA of irradiated cells, which can promote tumorigenesis. As malignant transformation is a process that takes many years, there are intermediate stages of cells that have initiated the process but have not yet evolved into cancer. The study here aimed to investigate the effect of ionizing radiation on normal and partially transformed human mammary epithelial cells. Breast primary epithelial cells were derived from normal breast tissue from two different donors and modified by transduction with the SV40 small and large T antigen and hTERT genes to obtain partially transformed cells and also with HRAS to completely and experimentally transform them. After exposure to different doses of ionizing radiation, oncogenic features were analyzed by means of an anchorage-independent growth assay and 3D cell culture. The addition of radiation exposure resulted in an increase in the number and size of colonies formed in each of the conditions analyzed and in the reduction of the capacity of partially transformed cells to form properly polarized 3D structures. Moreover, partially transformed cells require lower doses of radiation than healthy cells to enhance anchorage-independent growth capacity. Although cells from different donors have a different degree of sensitivity in the response to radiation, a higher sensitivity to the radiation-induced cell transformation process was observed in those cells that had already initiated the oncogenic process, which require higher doses of radiation to complete the transformation process. IMPLICATIONS: Individuals carrying accumulation of genetic alterations may have an increased susceptibility to radiation-induced neoplastic transformation.

MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer.

In thyroid cancers, MET receptor overexpression has been associated with higher risk of metastatic progression. In this study, it was shown that the anaplastic thyroid cancer (ATC)-derived TTA1 cell line overexpressed MET. By using FISH and relative quantification by qPCR, it was demonstrated that this overexpression resulted from a MET amplification with more than 20 copies. As expected, MET overexpression led to its constitutive activation and upregulated signaling towards the MAPK, PI3K/AKT, STAT3 and NF-κB pathways. Since the usual feature of MET-amplified cell lines is the "MET addiction" for their cell proliferation, the effect of the highly selective ATP competitive MET inhibitor PHA665752 was analyzed. While PHA665752 strongly inhibited the MAPK pathway, it did not reduce cell proliferation in TTA1 cells (IC50 = 4100 nM). This resistance to PHA665752 of the TTA1 cell line was demonstrated to be related to EGFR-MET functional cross-talk and PI3K/AKT and NF-κB signaling. Nevertheless, PHA665752 suppressed the anchorage-independent growth capacity of the TTA1 cell line and reduced cell migration and invasion in a transwell assay. The role of activated MET in these neoplastic properties of the TTA1 cells was also proved with si-MET-RNA targeting. Thus, this work highlights the TTA1 cell line as the first model of MET amplification in an ATC cell line, which leads to MET constitutive activation and underlies its neoplastic properties. Besides being a useful model for MET inhibitors screening, the TTA1 cell line also supports the argument for searching for MET amplification in ATC, as it could have therapeutic implications.

Development of a novel and economical agar-based non-adherent three-dimensional culture method for enrichment of cancer stem-like cells

BackgroundNon-adherent or ultra-low attachment three-dimensional (3D) culture, also called sphere formation assay, has been widely used to assess the malignant phenotype and stemness potential of transformed or cancer cells. This method is also popularly used to isolate the cancer stem-like cells (CSCs) or tumor-initiating cells based on their unique anchorage-independent growth or anoikis-resistant capacity. Different non-adhesive coating agents, such as poly-2-hydroxyethyl methacrylate (poly-HEMA) and synthetic hydrogels, have been used in this non-adherent 3D culture. However, preparation of non-adherent culture-ware is labor-intensive and technically demanding, and also costs of commercial non-adherent culture-ware prepared with various coating agents are relatively expensive and the culture-ware cannot be used repeatedly.MethodsIn this study, we developed a non-adherent 3D culture method based on agar coating for growing tumor spheres derived from various cancer cell lines and primary prostate cancer tissues under a non-adherent and serum-free condition. The tumor spheres generated by this 3D culture method were analyzed on their expression profiles of CSC-associated markers by reverse transcription quantitative polymerase chain reaction, presence and relative proportion of CSCs by fluorescence-activated cell sorting (CD133+/CD44+ cell sorting) and also a CSC-visualizing reporter system responsive to OCT4 and SOX2 (SORE6), and in vivo tumorigenicity. The repeated use of agar-coated plates for serial passages of tumor spheres was also evaluated.ResultsOur results validated that the multicellular tumor spheres generated by this culture method were enriched of CSCs, as evidenced by their enhanced expression profiles of CSC markers, presence of CD133+/CD44+ or SORE6+ cells, enhanced self-renewal capacity, and in vivo tumorigenicity, indicating its usefulness in isolation and enrichment of CSCs. The agar-coated plates could be used multiple times in serial passages of tumor spheres.ConclusionsThe described agar-based 3D culture method offers several advantages as compared with other methods in isolation of CSCs, including its simplicity and low-cost and repeated use of agar-coated plates for continuous passages of CSC-enriched spheres.

Practical exercises with the EFSA WEB app based on PROAST: Presentation of case studies and modelling of provided datasets

Aristolochic acid I (AAI) is a phytocompound that is linked to the progressive renal disease and development of human urothelial carcinoma. The bladder cancer-associated protein (BLCAP) gene exhibits a tumor suppressor function in various tumors, including bladder carcinoma. This study evaluated the effect of AAI on BLCAP expression and its associated mechanism in human cells. Administering AAI to human embryonic kidney cells (HEK293), human proximal tubule epithelial cells (HK-2) and urinary bladder cancer cells (HT-1376) significantly reduced the expression of BLCAP mRNA and protein. AAI also effectively suppressed the luciferase activities driven by BLCAP promoters of various lengths in HEK293 cells. AAI significantly reduced both activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities in reporter assays, but further point mutations revealed that Ap-1 and NF-κB binding sites on the BLCAP promoter were not AAI-responsive elements. Application of the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), reversed the decline of BLCAP expression that had been induced by AAI. However, AAI exposure did not alter hypermethylation of the BLCAP promoter, determined by methyl-specific polymerase chain reaction (PCR) and bisulfate sequencing. Knocking down BLCAP in HEK293 cell line enhanced the potential for cellular migration, invasion, and proliferation, along with the induction of a capacity for anchorage-independent growth. In conclusion, AAI down-regulated the expression of BLCAP gene and the deficiency in BLCAP expression contributed to the malignant transformation of human cells, implying that BLCAP may have a role in mediating AAI-associated carcinogenesis.

Genome-wide binding of transcription factor ZEB1 in triple-negative breast cancer cells.

Zinc finger E‐box binding homeobox 1 (ZEB1) is a transcriptional regulator involved in embryonic development and cancer progression. ZEB1 induces epithelial‐mesenchymal transition (EMT). Triple–negative human breast cancers express high ZEB1 mRNA levels and exhibit features of EMT. In the human triple–negative breast cancer cell model Hs578T, ZEB1 associates with almost 2,000 genes, representing many cellular functions, including cell polarity regulation (DLG2 and FAT3). By introducing a CRISPR‐Cas9‐mediated 30 bp deletion into the ZEB1 second exon, we observed reduced migratory and anchorage‐independent growth capacity of these tumor cells. Transcriptomic analysis of control and ZEB1 knockout cells, revealed 1,372 differentially expressed genes. The TIMP metallopeptidase inhibitor 3 and the teneurin transmembrane protein 2 genes showed increased expression upon loss of ZEB1, possibly mediating pro‐tumorigenic actions of ZEB1. This work provides a resource for regulators of cancer progression that function under the transcriptional control of ZEB1. The data confirm that removing a single EMT transcription factor, such as ZEB1, is not sufficient for reverting the triple–negative mesenchymal breast cancer cells into more differentiated, epithelial‐like clones, but can reduce tumorigenic potential, suggesting that not all pro‐tumorigenic actions of ZEB1 are linked to the EMT.

Aristolochic acid I interferes with the expression of BLCAP tumor suppressor gene in human cells

Aristolochic acid I (AAI) is a phytocompound that is linked to the progressive renal disease and development of human urothelial carcinoma. The bladder cancer-associated protein (BLCAP) gene exhibits a tumor suppressor function in various tumors, including bladder carcinoma. This study evaluated the effect of AAI on BLCAP expression and its associated mechanism in human cells. Administering AAI to human embryonic kidney cells (HEK293), human proximal tubule epithelial cells (HK-2) and urinary bladder cancer cells (HT-1376) significantly reduced the expression of BLCAP mRNA and protein. AAI also effectively suppressed the luciferase activities driven by BLCAP promoters of various lengths in HEK293 cells. AAI significantly reduced both activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities in reporter assays, but further point mutations revealed that Ap-1 and NF-κB binding sites on the BLCAP promoter were not AAI-responsive elements. Application of the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), reversed the decline of BLCAP expression that had been induced by AAI. However, AAI exposure did not alter hypermethylation of the BLCAP promoter, determined by methyl-specific polymerase chain reaction (PCR) and bisulfate sequencing. Knocking down BLCAP in HEK293 cell line enhanced the potential for cellular migration, invasion, and proliferation, along with the induction of a capacity for anchorage-independent growth. In conclusion, AAI down-regulated the expression of BLCAP gene and the deficiency in BLCAP expression contributed to the malignant transformation of human cells, implying that BLCAP may have a role in mediating AAI-associated carcinogenesis.

Mek activity is required for ErbB2 expression in breast cancer cells detached from the extracellular matrix.

Detachment of non-malignant epithelial cells from the extracellullar matrix (ECM) triggers their growth arrest and apoptosis. Conversely, carcinoma cells can grow without adhesion to the ECM. This capacity for anchorage-independent growth is thought to be critical for tumor progression. ErbB2/Her2 oncoprotein is overproduced by a significant fraction of breast cancers and promotes anchorage-independent tumor cell growth by poorly understood mechanisms. In an effort to understand them we found that in order to produce ErbB2, detached breast cancer cells require the activity of an ErbB2 effector protein kinase Mek and that Mek-driven ErbB2 expression is neccesary for anchorage-independent growth of such cells. We observed that Mek inhibition does not alter ErbB2 mRNA levels in detached cancer cells and that ErbB2 protein loss induced by this inhibition can be blocked by a lysosomal inhibitor. We also noticed that an increase of the density of cancer cells detached from the ECM downregulates a Mek effector protein kinase Erk and causes ErbB2 loss. Those cells that survive after ErbB2 loss display resistance to trastuzumab, an anti-ErbB2 antibody used for ErbB2-positive breast cancer treatment. Thus, Mek-induced ErbB2 stabilization in detached breast cancer cells is critical for their ability to grow anchorage-independently and their trastuzumab sensitivity.