Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the `mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes. Oncogene (2012) 31, 1896-1909; doi:10.1038/onc.2011.378; published online 29 August 2011

A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors

Higher Notch signaling is known to be associated with hematological and solid cancers. We developed a potential immunotherapeutic monoclonal antibody (MAb) specific for the Negative Regulatory Region of Notch1 (NRR). The MAb604.107 exhibited higher affinity for the ``Gain-offunction'' mutants of Notch1 NRR associated with T Acute lymphoblastic Leukemia (T-ALL). Modeling of the mutant NRR with 12 amino-acid insertion demonstrated ``opening'' resulting in exposure of the S2-cleavage site leading to activated Notch1 signaling. The MAb, at low concentrations (1-2 mu g/ml), inhibited elevated ligand-independent Notch1 signaling of NRR mutants, augmented effect of Thapsigargin, an inhibitor of mutant Notch1, but had no effect on the wild-type Notch1. The antibody decreased proliferation of the primary T-ALL cells and depleted leukemia initiating CD34/CD44 high population. At relatively high concentrations, (10-20 mu g/ml), the MAb affected Notch1 signaling in the breast and colon cancer cell lines. The Notch-high cells sorted from solid-tumor cell lines exhibited characteristics of cancer stem cells, which were inhibited by the MAb. The antibody also increased the sensitivity to Doxorubucinirubicin. Further, the MAb impeded the growth of xenografts from breast and colon cancer cells potentiated regression of the tumors along with Doxorubucin. Thus, this antibody is potential immunotherapeutic tool for different cancers.

The determination of stem cell fate by 3D scaffold structures through the control of cell shape

Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage. Published by Elsevier Ltd.

Inflammation and cancer stem cells

Cancer stem cells are becoming recognised as being responsible for metastasis and treatment resistance. The complex cellular and molecular network that regulates cancer stem cells and the role that inflammation plays in cancer progression are slowly being elucidated. Cytokines, secreted by tumour associated immune cells, activate the necessary pathways required by cancer stem cells to facilitate cancer stem cells progressing through the epithelial-mesenchymal transition and migrating to distant sites. Once in situ, these cancer stem cells can secrete their own attractants, thus providing an environment whereby these cells can continue to propagate the tumour in a secondary niche. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Competing views on cancer

Despite intense research efforts that have provided enormous insight, cancer continues to be a poorly understood disease. There has been much debate over whether the cancerous state can be said to originate in a single cell or whether it is a reflection of aberrant behaviour on the part of a `society of cells'. This article presents, in the form of a debate conducted among the authors, three views of how the problem might be addressed. We do not claim that the views exhaust all possibilities. These views are (a) the tissue organization field theory (TUFT) that is based on a breakdown of tissue organization involving many cells from different embryological layers, (b) the cancer stem cell (CSC) hypothesis that focuses on genetic and epigenetic changes that take place within single cells, and (c) the proposition that rewiring of the cell's protein interaction networks mediated by intrinsically disordered proteins (IDPs) drives the tumorigenic process. The views are based on different philosophical approaches. In detail, they differ on some points and agree on others. It is left to the reader to decide whether one approach to understanding cancer appears more promising than the other.

Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates

In the context of the role of multiple physical factors in dictating stem cell fate, the present paper demonstrates the effectiveness of the intermittently delivered external electric field stimulation towards switching the stem cell fate to specific lineage, when cultured in the absence of biochemical growth factors. In particular, our findings present the ability of human mesenchymal stem cells (hMSCs) to respond to the electric stimuli by adopting extended neural-like morphology on conducting polymeric substrates. Polyaniline (PANI) is selected as the model system to demonstrate this effect, as the electrical conductivity of the polymeric substrates can be systematically tailored over a broad range (10(-9) to 10 S/cm) from highly insulating to conducting by doping with varying concentrations (10(-5) to 1 M) of HCl. On the basis of the culture protocol involving the systematic delivery of intermittent electric field (dc) stimulation, the parametric window of substrate conductivity and electric field strength was established to promote significant morphological extensions, with minimal cellular damage. A time dependent morphological change in hMSCs with significant filopodial elongation was observed after 7 days of electrically stimulated culture. Concomitant with morphological changes, a commensurate increase in the expression of neural lineage commitment markers such as nestin and PI tubulin was recorded from hMSCs grown on highly conducting substrates, as revealed from the mRNA expression analysis using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) as well as by immune-fluorescence imaging. Therefore, the present work establishes the key role of intermittent and systematic delivery of electric stimuli as guidance cues in promoting neural-like differentiation of hMSCs, when grown on electroconductive substrates. (C) 2014 Elsevier Ltd. All rights reserved.

Chemical Functionalization of Graphene To Augment Stem Cell Osteogenesis and Inhibit Biofilm Formation on Polymer Composites for Orthopedic Applications

Toward designing the next generation of resorbable biomaterials for orthopedic applications, we studied poly(epsilon-caprolactone) (PCL) composites containing graphene. The role, if any, of the functionalization of graphene on mechanical properties, stem cell response, and biofilm formation was systematically evaluated. PCL composites of graphene oxide (GO), reduced GO (RGO), and amine-functionalized GO (AGO) were prepared at different filler contents (1%, 3%, and 5%). Although the addition of the nanoparticles to PCL markedly increased the storage modulus, this increase was largest for GO followed by AGO and RGO. In vitro cell studies revealed that the AGO and GO particles significantly increased human mesenchymal stem cell proliferation. AGO was most effective in augmenting stem cell osteogenesis leading to mineralization. Bacterial studies revealed that interaction with functionalized GO induced bacterial cell death because of membrane damage, which was further accentuated by amine groups in AGO. As a result, AGO composites were best at inhibiting biofilm formation. The synergistic effect of oxygen containing functional groups and amine groups on AGO imparts the optimal combination of improved modulus, favorable stem cell response, and biofilm inhibition in AGO-reinforced composites desired for orthopedic applications. This work elucidates the importance of chemical functionalization of graphene in polymer composites for biomedical applications.

Magnetic field assisted stem cell differentiation - role of substrate magnetization in osteogenesis

Among the multiple modulatory physical cues explored to regulate cellular processes, the potential of magneto-responsive substrates in magnetic field stimulated stem cell differentiation is still unperceived. In this regard, the present work demonstrates how an external magnetic field can be applied to direct stem cell differentiation towards osteogenic commitment. A new culture methodology involving periodic delivery of 100 mT static magnetic field (SMF) in combination with HA-Fe3O4 magnetic substrates possessing a varying degree of substrate magnetization was designed for the study. The results demonstrate that an appropriate combination of weakly ferromagnetic substrates and SMF exposure enhanced cell viability, DNA synthesis and caused an early switchover to osteogenic lineage as supported by Runx2 immunocytochemistry and ALP expression. However, the mRNA expression profile of early osteogenic markers (Runx2, ALP, Col IA) was comparable despite varying substrate magnetic properties (diamagnetic to ferromagnetic). On the contrary, a remarkable upregulation of late bone development markers (OCN and OPN) was explicitly detected on weak and strongly ferromagnetic substrates. Furthermore, SMF induced matrix mineralization with elevated calcium deposition on similar substrates, even in the absence of osteogenic supplements. More specifically, the role of SMF in increasing intracellular calcium levels and in inducing cell cycle arrest at G0/G1 phase was elucidated as the major molecular event triggering osteogenic differentiation. Taken together, the above results demonstrate the competence of magnetic stimuli in combination with magneto-responsive biomaterials as a potential strategy for stem cell based bone tissue engineering.

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog

Background: The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells. Methods: Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro-and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response. Results: We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition ( EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NF kappa B promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NF kappa B pathway whereas Bmi1 knockdown reduced the expression of NF kappa B target genes, suggesting that Bmi1 might regulate Nanog expression through the NF kappa B pathway. Conclusions: Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NF kappa B pathway.

RAD51 as a potential biomarker and therapeutic target for pancreatic cancer

Chemotherapy is a very important therapeutic strategy for cancer treatment. The failure of conventional and molecularly targeted chemotherapeutic regimes for the treatment of pancreatic cancer highlights a desperate need for novel therapeutic interventions. Chemotherapy often fails to eliminate all tumor cells because of intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Overexpression of RAD51 protein, a key player in DNA repair/recombination has been observed in many cancer cells and its hyperexpression is implicated in drug resistance. Recent studies suggest that RAD51 overexpression contributes to the development, progression and drug resistance of pancreatic cancer cells. Here we provide a brief overview of the available pieces of evidence in support of the role of RAD51 in pancreatic tumorigenesis and drug resistance, and hypothesize that RAD51 could serve as a potential biomarker for diagnosis of pancreatic cancer. We discuss the possible involvement of RAD51 in the drug resistance associated with epithelial to mesenchymal transition and with cancer stem cells. Finally, we speculate that targeting RAD51 in pancreatic cancer cells may be a novel approach for the treatment of pancreatic cancer. (C) 2011 Elsevier B.V. All rights reserved.