Acquisition Of Stem Cell Characteristics Research Articles

In Vitro Characterization of Reversine-Treated Gingival Fibroblasts and Their Safety Evaluation after In Vivo Transplantation.

Reversine is a purine derivative that has been investigated with regard to its biological effects, such as its anticancer properties and, mostly, its ability to induce the dedifferentiation of adult cells, increasing their plasticity. The obtained dedifferentiated cells have a high potential for use in regenerative procedures, such as regenerative dentistry (RD). Instead of replacing the lost or damaged oral tissues with synthetic materials, RD uses stem cells combined with matrices and an appropriate microenvironment to achieve tissue regeneration. However, the currently available stem cell sources present limitations, thus restricting the potential of RD. Based on this problem, new sources of stem cells are fundamental. This work aims to characterize mouse gingival fibroblasts (GFs) after dedifferentiation with reversine. Different administration protocols were tested, and the cells obtained were evaluated regarding their cell metabolism, protein and DNA contents, cell cycle changes, morphology, cell death, genotoxicity, and acquisition of stem cell characteristics. Additionally, their teratoma potential was evaluated after in vivo transplantation. Reversine caused toxicity at higher concentrations, with decreased cell metabolic activity and protein content. The cells obtained displayed polyploidy, a cycle arrest in the G2/M phase, and showed an enlarged size. Additionally, apoptosis and genotoxicity were found at higher reversine concentrations. A subpopulation of the GFs possessed stem properties, as supported by the increased expression of CD90, CD105, and TERT, the existence of a CD106+ population, and their trilineage differentiation capacity. The dedifferentiated cells did not induce teratoma formation. The extensive characterization performed shows that significant functional, morphological, and genetic changes occur during the dedifferentiation process. The dedifferentiated cells have some stem-like characteristics, which are of interest for RD.

Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

BackgroundTriple-negative breast cancers (TNBC) have a relatively poor prognosis and responses to targeted therapies. Between 25 and 39% of TNBCs are claudin-low, a poorly differentiated subtype enriched for mesenchymal, stem cell and mitogen-activated signaling pathways. We investigated the role of the cell-surface co-receptor NRP1 in the biology of claudin-low TNBC.MethodsThe clinical prognostic value of NRP1 was determined by Kaplan–Meier analysis. GSVA analysis of METABRIC and Oslo2 transcriptomics datasets was used to correlate NRP1 expression with claudin-low gene signature scores. NRP1 siRNA knockdown was performed in MDA-MB-231, BT-549, SUM159 and Hs578T claudin-low cells and proliferation and viability measured by live cell imaging and DNA quantification. In SUM159 orthotopic xenograft models using NSG mice, NRP1 was suppressed by shRNA knockdown or systemic treatment with the NRP1-targeted monoclonal antibody Vesencumab. NRP1-mediated signaling pathways were interrogated by protein array and Western blotting.ResultsHigh NRP1 expression was associated with shorter relapse- and metastasis-free survival specifically in ER-negative BrCa cohorts. NRP1 was over-expressed specifically in claudin-low clinical samples and cell lines, and NRP1 knockdown reduced proliferation of claudin-low cells and prolonged survival in a claudin-low orthotopic xenograft model. NRP1 inhibition suppressed expression of the mesenchymal and stem cell markers ZEB1 and ITGA6, respectively, compromised spheroid-initiating capacity and exerted potent anti-tumor effects on claudin-low orthotopic xenografts (12.8-fold reduction in endpoint tumor volume). NRP1 was required to maintain maximal RAS/MAPK signaling via EGFR and PDGFR, a hallmark of claudin-low tumors.ConclusionsThese data implicate NRP1 in the aggressive phenotype of claudin-low breast cancer and offer a novel targeted therapeutic approach to this poor prognosis subtype.

Abstract PO-10: Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture

Abstract Linker histone H1 binds to nucleosomes and causes chromatin compaction, yet little is known about their biologic function. Somatic missense mutations in histone 1 genes (HIST1H1B-E) occur in ~30% of follicular lymphomas and DLBCL, with significant mutual co-occurrence among these alleles, most frequently involving H1C and H1E. Examining whole exomes in 547 DLBCL patients, we found significant enrichment for H1 gene SNVs and copy number loss in the MCD class of ABC-DLBCLs as compared to other subtypes. Next we performed a genetic driver analysis in 101 DLBCLs with matched germline control to identify mutations significantly enriched over background somatic variation. We find that lymphoma-associated H1 alleles H1C and H1E are true genetic driver mutations in lymphomas. Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to bind to chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRAP assays in live cells. Hence both types of mutation confer loss of function. Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T cell-dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments, indicating that they have superior fitness (p=0.0086). We performed RNA-seq in H1C/EDKO GC B-cells, which revealed an aberrant gene derepression signature. Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming and are normally silenced during early development by the PRC2 complex (p <0.05 to 1e-05). Indeed, histone mass spectrometry showed reduced H3K27me3 (p=0.0003) and increased H3K36me2 (p =0.001) in H1C/EDKO GC B cells. This prompted us to characterize the epigenome of purified H1C/EDKO vs WT GC B cells using Hi-C, ATAC-seq, and ChIP-seq for multiple histone marks. H1 deficiency causes profound architectural remodeling of the genome characterized by large-scale yet focal compartment shifts from compartment B to compartment A. The degree of decompaction results in distinct epigenetic states, primarily due to gain of H3K36me2 and/or loss of repressive H3K27me3. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR<0.001) in the RNA-seq profiles of independent cohorts of DLBCL patients with H1C and H1E mutations. We crossed H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Consistent with acquisition of stem cell characteristics, H1C+/-H1E+/-;VavP-Bcl2 but not VavP-Bcl2 primary lymphoma cells manifested lymphoma-initiating functionality after secondary transplantation into recipient animals. Citation Format: Nevin Z. Yusufova, Matt Teater, Andreas Kloetgen, Alexey Soshnev, Adewola Osunade, Christopher Chin, Ashley Doane, Louis Staudt, David Scott, Neil Kelleher, Aristotelis Tsirigos, Marcin Imielinski, Yael David, David Allis, Ethel Cesarman, Ari Melnick. Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-10.

Histone 1 Mutations Drive Lymphomagenesis By Inducing Primitive Stem Cell Functions and Epigenetic Instructions through Profound 3D Re-Organization of the B-Cell Genome

Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.

Long non‑coding RNA00887 reduces the invasion and metastasis of non‑small cell lung cancer by causing the degradation of miRNAs.

Long non‑coding RNAs (lncRNAs) can act as carcinogenic or cancer suppressive factors during the pathogenesis, invasion and metastasis of non‑small cell lung cancer (NSCLC). The current study explored the role of long intergenic non‑protein coding RNA 00887 (LINC00887) and competing endogenous RNAs (ceRNAs). It was revealed that LINC00887 interacts with several microRNAs (miRs), which regulates downstream genes such as fibronectin1, MET proto‑oncogene, receptor tyrosine kinase and mothers against decapentaplegic homolog 4, which are associated with the spread of lung cancer. The experimental results also suggested that LINC00887 can stimulate miR‑613, miR‑206 and miR‑1‑2 to become competing endogenous RNAs, which may regulate the epithelial‑mesenchymal transition of NSCLC cells through the transforming growth factor‑â signal transduction pathway, and therefore promote the migration of cells and the acquisition of stem cell characteristics. Therefore, it can be concluded that high levels of LINC00887 can accelerate the malignant transformation ability of NSCLC cells.

Abstract P1-07-01: Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties

Abstract Introduction: Breast cancer is the most commonly diagnosed cancer and third-leading cause of cancer-related deaths in women in America. A quarter of lesions diagnosed annually are ductal carcinoma in situ (DCIS), one of the earliest pre-invasive forms of invasive ductal carcinoma (IDC). Without therapeutic intervention, 30-50% of DCIS cases can progress to IDC. Understanding the mechanisms regulating progression from DCIS to IDC would help identify biomarkers to stratify DCIS patients at higher risk of progression or recurrence. Cumulative literature suggests the earliest phase of dissemination from the primary tumor is driven by the epithelial-mesenchymal transition (EMT) program. DEAR1 is a tumor suppressor gene which is mutated, undergoes loss of heterozygosity in breast cancer, and is downregulated in DCIS lesions. DEAR1 regulates acinar morphogenesis and cell polarity, and is a negative regulator of TGF-beta-driven EMT by binding to and ubiquitinating SMAD3, thereby limiting the amount of SMAD3 available to activate an EMT signature. Overexpression of EMT master regulators, or exposure to TGF-beta in immortalized human mammary epithelial cells (HMECs), results in mammosphere formation and breast stem cell markers, thus linking the EMT process to acquisition of stem cell characteristics. Methods: Stable lentiviral shRNA knockdown, in vitro mammosphere assay, cytospin, immunofluorescence, immunoprecipitation, western blot analysis, ubiquitination and real-time quantitative PCR were performed. Results: DEAR1 knockdown in immortal HMECs resulted in a significant enhancement of primary mammosphere formation and growth compared to controls, suggesting that DEAR1 may regulate stem/progenitor cell properties; this effect was greater when cells were exposed to TGF-beta. To determine if DEAR1 regulates stem cell properties through regulation of SMAD3 levels, DEAR1-SMAD3 double knockdown clones were examined for mammosphere formation. Results indicated fewer mammospheres in double knockdown clones, but only in the presence of TGF-beta, suggesting that the mammosphere phenotype is partially dependent on the TGF-beta-SMAD3 pathway. The observation that loss of DEAR1 alone results in a stem/progenitor cell phenotype indicates that DEAR1 may regulate mammosphere formation independent of its role in regulating the TGF-beta pathway. HMEC mammospheres express higher levels of stem cell marker aldehyde dehydrogenase (ALDH1) and co-express luminal and basal cytokeratins, suggesting bipotential capacity. We also observed upregulation of SNAI2 and ZEB1, master EMT and stem cell regulators, in DEAR1 knockdown HMECs. DEAR1 interacted with SNAI2 by co-immunoprecipitation analysis and also promoted the polyubiquitination of SNAI2. Conclusions: Loss of the tumor suppressor/polarity regulator DEAR1 promotes stem cell properties in part through the DEAR1-TGF-beta-SMAD3 axis. We demonstrate a DEAR1-SNAI2 axis that may partially regulate stem/progenitor cell properties in HMECs through the polyubiquitination of SNAI2, a master regulator of EMT and stemness. These mechanisms governing acquisition of the stem cell properties may contribute to understanding how DCIS progress to IDC. Citation Format: Le U, Chen N, Balasenthil S, Killary AM. Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-07-01.

SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium.

The cell of origin of colon cancer is typically thought to be the resident somatic stem cells, which are immortal and escape the continual cellular turnover characteristic of the intestinal epithelium. However, recent studies have identified certain conditions in which differentiated cells can acquire stem-like properties and give rise to tumors. Defining the origins of tumors will inform cancer prevention efforts as well as cancer therapies, as cancers with distinct origins often respond differently to treatments. We report here a new condition in which tumors arise from the differentiated intestinal epithelium. Inactivation of the differentiation-promoting transcription factor SMAD4 in the intestinal epithelium was surprisingly well tolerated in the short term. However, after several months, adenomas developed with characteristics of activated WNT signaling. Simultaneous loss of SMAD4 and activation of the WNT pathway led to dedifferentiation and rapid adenoma formation in differentiated tissue. Transcriptional profiling revealed acquisition of stem cell characteristics, and colabeling indicated that cells expressing differentiated enterocyte markers entered the cell cycle and reexpressed stem cell genes upon simultaneous loss of SMAD4 and activation of the WNT pathway. These results indicate that SMAD4 functions to maintain differentiated enterocytes in the presence of oncogenic WNT signaling, thus preventing dedifferentiation and tumor formation in the differentiated intestinal epithelium.Significance: This work identifies a mechanism through which differentiated cells prevent tumor formation by suppressing oncogenic plasticity. Cancer Res; 78(17); 4878-90. ©2018 AACR.

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study.

BackgroundBreast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial–mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes.MethodsA total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence.ResultsThe isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1+) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44+CD24− BCSCs from MCF-7 cells.DiscussionThis study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs.

Chronic oxidative stress leads to malignant transformation along with acquisition of stem cell characteristics, and epithelial to mesenchymal transition in human renal epithelial cells.

Oxidative injury to cellular macromolecules has been suggested as a common pathway shared by multiple etiological factors for kidney cancer. Whether the chronic oxidative stress alone is sufficient to induce malignant transformation in human kidney cells is not clear. Therefore, the objective of this study was to evaluate the effect of H2O2-induced chronic oxidative stress on growth, and malignant transformation of HK-2 normal kidney epithelial cells. This study revealed that chronic oxidative stress causes increased growth and neoplastic transformation in normal kidney epithelial cells at non-cytotoxic dose and increased adaptation to cytotoxic level. This was confirmed by gene expression changes, cell cycle analysis, anchorage independent growth assay and in vivo tumorigenicity in nude mice. Stem cells characteristics as revealed by up-regulation of stem cell marker genes, and morphological changes indicative of EMT with up regulation of mesenchymal markers were also observed in cells exposed to chronic oxidative stress. Antioxidant NAC did not reverse the chronic oxidative stress-induced growth, and adaptation suggesting that perturbed biological function in these cells are permanent. Partial reversal of oxidative stress-induced growth, and adaptation by silencing of Oct 4 and Snail1, respectively, suggest that these changes are mediated by acquisition of stem cell and EMT characteristics. In summary, this study for the first time suggests that chronic exposure to elevated levels of oxidative stress is sufficient to induce malignant transformation in kidney epithelial cells through acquisition of stem cell characteristics. Additionally, the EMT plays an important role in increased adaptive response of renal cells to oxidative stress.

Androgen receptor splice variants contribute to prostate cancer aggressiveness through induction of EMT and expression of stem cell marker genes.

The mechanism(s) by which androgen receptor (AR) splice variants contribute to castration-resistant prostate cancer (CRPC) is still lacking. Expressions of epithelial-to-mesenchymal transition (EMT) and stem cell markers were molecularly tested using prostate cancer (PCa) cells transfected with AR and AR3 (also known as AR-V7) plasmids or siRNA, and also cultured cells under androgen deprivation therapy (ADT) condition. Cell migration, clonogenicity, sphere-forming capacity was assessed using PCa cells under all experimental conditions and 3,3'-diindolylmethane (DIM; BR-DIM) treatment. Human PCa samples from BR-DIM untreated or treated patients were also used for assessing the expression of AR3 and stem cell markers. Overexpression of AR led to the induction of EMT phenotype, while overexpression of AR3 not only induced EMT but also led to the expression of stem cell signature genes. More importantly, ADT enhanced the expression of AR and AR3 concomitant with up-regulated expression of EMT and stem cell marker genes. Dihydrotestosterone (DHT) treatment decreased the expression of AR and AR3, and reversed the expression of these EMT and stem cell marker genes. BR-DIM administered to PCa patients prior to radical prostatectomy inhibited the expression of cancer stem cell markers consistent with inhibition of self-renewal of PCa cells after BR-DIM treatment. AR variants could contribute to PCa progression through induction of EMT and acquisition of stem cell characteristics, which could be attenuated by BR-DIM, suggesting that BR-DIM could become a promising agent for the prevention of CRPC and/or for the treatment of PCa.

Direct reprogramming by oncogenic Ras and Myc

Genetically or epigenetically defined reprogramming is a hallmark of cancer cells. However, a causal association between genome reprogramming and cancer has not yet been conclusively established. In particular, little is known about the mechanisms that underlie metastasis of cancer, and even less is known about the identity of metastasizing cancer cells. In this study, we used a model of conditional expression of oncogenic KrasG12D allele in primary mouse cells to show that reprogramming and dedifferentiation is a fundamental early step in malignant transformation and cancer initiation. Our data indicate that stable expression of activated KrasG12D confers on cells a large degree of phenotypic plasticity that predisposes them to neoplastic transformation and acquisition of stem cell characteristics. We have developed a genetically tractable model system to investigate the origins and evolution of metastatic pancreatic cancer cells. We show that metastatic conversion of KrasG12D-expressing cells that exhibit different degrees of differentiation and malignancy can be reconstructed in cell culture, and that the proto-oncogene c-Myc controls the generation of self-renewing metastatic cancer cells. Collectively, our results support a model wherein non-stem cancer cells have the potential to dedifferentiate and acquire stem cell properties as a direct consequence of oncogene-induced plasticity. Moreover, the disturbance in the normally existing dynamic equilibrium between cancer stem cells and non-stem cancer cells allows the formation of cancer stem cells with high metastatic capacity at any time during cancer progression.

Canine Mammary Cancer Stem Cells are Radio- and Chemo- Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype.

Canine mammary carcinoma is the most common cancer among female dogs and is often fatal due to the development of distant metastases. In humans, solid tumors are made up of heterogeneous cell populations, which perform different roles in the tumor economy. A small subset of tumor cells can hold or acquire stem cell characteristics, enabling them to drive tumor growth, recurrence and metastasis. In veterinary medicine, the molecular drivers of canine mammary carcinoma are as yet undefined. Here we report that putative cancer stem cells (CSCs) can be isolated form a canine mammary carcinoma cell line, REM134. We show that these cells have an increased ability to form tumorspheres, a characteristic of stem cells, and that they express embryonic stem cell markers associated with pluripotency. Moreover, canine CSCs are relatively resistant to the cytotoxic effects of common chemotherapeutic drugs and ionizing radiation, indicating that failure of clinical therapy to eradicate canine mammary cancer may be due to the survival of CSCs. The epithelial to mesenchymal transition (EMT) has been associated with cancer invasion, metastasis, and the acquisition of stem cell characteristics. Our results show that canine CSCs predominantly express mesenchymal markers and are more invasive than parental cells, indicating that these cells have a mesenchymal phenotype. Furthermore, we show that canine mammary cancer cells can be induced to undergo EMT by TGFβ and that these cells have an increased ability to form tumorspheres. Our findings indicate that EMT induction can enrich for cells with CSC properties, and provide further insight into canine CSC biology.