Acquisition Of Stem Cell-like Properties Research Articles

A bioactive composite scaffold enhances osteochondral repair by using thermosensitive chitosan hydrogel and endothelial lineage cell-derived chondrogenic cell

Articular cartilage regeneration is a major challenge in orthopedic medicine. Endothelial progenitor cells (EPCs) are a promising cell source for regenerative medicine applications. However, their roles and functions in cartilage regeneration are not well understood. Additionally, thermosensitive chitosan hydrogels have been widely used in tissue engineering, but further development of these hydrogels incorporating vascular lineage cells for cartilage repair is insufficient. Thus, this study aimed to characterize the ability of EPCs to undergo endothelial–mesenchymal stem cell transdifferentiation and chondrogenic differentiation and investigate the ability of chondrogenic EPC-seeded thermosensitive chitosan-graft-poly (N-isopropylacrylamide) (CEPC-CSPN) scaffolds to improve healing in a rabbit osteochondral defect (OCD) model. EPCs were isolated and endothelial-to-mesenchymal transition (EndMT) was induced by transforming growth factor-β1 (TGF-β1); these EPCs are subsequently termed transdifferentiated EPCs (tEPCs). The stem cell-like properties and chondrogenic potential of tEPCs were evaluated by a series of in vitro assays. Furthermore, the effect of CEPC-CSPN scaffolds on OCD repair was evaluated. Our in vitro results confirmed that treatment of EPC with TGF-β1 induced EndMT and the acquisition of stem cell-like properties, producing tEPCs. Upon inducing chondrogenic differentiation of tEPCs (CEPCs), the cells exhibited significantly enhanced chondrogenesis and chondrocyte surface markers after 25 days. The TGF-β1-induced differentiation of EPCs is mediated by both the TGF-β/Smad and extracellular signal-regulated kinase (Erk) pathways. The CEPC-CSPN scaffold reconstructed well-integrated translucent cartilage and repaired subchondral bone in vivo, exhibiting regenerative capacity. Collectively, our results suggest that the CEPC-CSPN scaffold induces OCD repair, representing a promising approach to articular cartilage regeneration.

Helicobacter pylori CagA protein induces gastric cancer stem cell-like properties through the Akt/FOXO3a axis.

The presence of Helicobacter pylori (H. pylori) infection poses a substantial risk for the development of gastric adenocarcinoma. The primary mechanism through which H. pylori exerts its bacterial virulence is the cytotoxin CagA. This cytotoxin has the potential to induce inter-epithelial mesenchymal transition, proliferation, metastasis, and the acquisition of stem cell-like properties in gastric cancer (GC) cells infected with CagA-positive H. pylori. Cancer stem cells (CSCs) represent a distinct population of cells capable of self-renewal and generating heterogeneous tumor cells. Despite evidence showing that CagA can induce CSCs-like characteristics in GC cells, the precise mechanism through which CagA triggers the development of GC stem cells (GCSCs) remains uncertain. This study reveals that CagA-positive GC cells infected with H. pylori exhibit CSCs-like properties, such as heightened expression of CD44, a specific surface marker for CSCs, and increased ability to form tumor spheroids. Furthermore, we have observed that H. pylori activates the PI3K/Akt signaling pathway in a CagA-dependent manner, and our findings suggest that this activation is associated with the CSCs-like characteristics induced by H. pylori. The cytotoxin CagA, which is released during H. pylori infection, triggers the activation of the PI3K/Akt signaling pathway in a CagA-dependent manner. Additionally, CagA inhibits the transcription of FOXO3a and relocates it from the nucleus to the cytoplasm by activating the PI3K/Akt pathway. Furthermore, the regulatory function of the Akt/FOXO3a axis in the transformation of GC cells into a stemness state was successfully demonstrated.

Abstract A019: Epigenetic regulation by H3K9 methylation enables resistance to anti-androgen therapy in prostate cancer

Abstract Prostate cancer (PCa) is a major cause of cancer mortality in the United States. Androgen receptor (AR) is the key transcriptional regulator for the development and progression of prostate cancer. Androgen deprivation therapy (ADT) with medical castration and treatment with second-generation anti-androgens such as enzalutamide and abiraterone acetate are important treatments for advanced prostate cancer. Previous studies have shown resistance to enzalutamide develops through various alternative molecular mechanisms including AR gene amplification, mutations in the ligand binding domains of AR, and increased expression of glucocorticoid receptor (GR). Recent reports further indicate that resistance to enzalutamide is accompanied with various de novo genomic and transcriptional changes that lead to reduced AR activity and acquisition of stem-cell like properties along with increased expression of genes associated with epithelial-to-mesenchymal transition and inflammatory signaling pathway, including interleukin 6 (IL6), janus kinase-signal transducer and activator of transcription (JAK-STAT) and interferon (IFN). In our study, we have unraveled a novel mechanism of epigenetic regulation mediated by H3K9 methylation in response to anti-androgen treatment in PCa cells. We have shown enzalutamide treatment leads to aberrant activation of endogenous retroelements (REs) which causes accumulation of double stranded RNA (dsRNA) in the PCa cells. This results in an antiviral response mediated via IFN signaling and induction of apoptosis, a phenomenon known as viral mimicry. Using a forward genetic screen, we have identified that the epigenetic machinery associated with H3K9 methylation is responsible for suppressing the aberrant transcription of REs in response to anti-androgen treatment. Further, overexpression of the terminal H3K9me3 writers lead to the suppression of REs whereas pharmacological or genetic inhibition of H3K9 trimethylation writers and readers restored REs expression, abrogating resistance to anti-androgen. Moreover, elevated expression of terminal H3K9me3 writers is associated with poor outcomes in patients subjected to hormonal therapy. Interestingly, we have observed that inhibition of double-stranded RNA-specific adenosine deaminase (ADAR1) leads to accumulation of dsRNA and modulates GR expression in PCa cells. Taken together, our study highlights the role of H3K9 trimethylation in mediating resistance to anti-androgen therapy in prostate cancer. Notably, members of the H3K9 trimethylation machinery could potentially serve as therapeutic targets and combinatorial approaches targeting epigenetic regulatory enzymes as well as androgen signaling axis could be a promising strategy for overcoming drug resistance and immune evasion in prostate cancer. Citation Format: Ritika Tiwari, Mehdi Baratchian, Michael Berk, Jianneng Li, Amy Guerinot, Vladimir Makarov, Timothy A. Chan, Abhishek A. Chakraborty, Nima Sharifi. Epigenetic regulation by H3K9 methylation enables resistance to anti-androgen therapy in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A019.

Concise review: Cancer cell reprogramming and therapeutic implications

The cancer stem cell (CSC) hypothesis postulates that cancer originates from the malignant transformation of stem cells and is considered to apply to a variety of cancers. Additionally, cancer cells alter metabolic processes to sustain their characteristic uncontrolled growth and proliferation. Further, microRNAs (miRNAs) are found to be involved in acquisition of stem cell-like properties, regulation and reprogramming of cancer cells during cancer progression through its post-transcriptional-regulatory activity. In this concise review, we aim to integrate the current knowledge and recent advances to elucidate the mechanisms involved in the regulation of cell reprogramming and highlights the potential therapeutic implications for the future.

Brain Microvascular Pericytes in Vascular Cognitive Impairment and Dementia.

Pericytes are unique, multi-functional mural cells localized at the abluminal side of the perivascular space in microvessels. Originally discovered in 19th century, pericytes had drawn less attention until decades ago mainly due to lack of specific markers. Recently, however, a growing body of evidence has revealed that pericytes play various important roles: development and maintenance of blood–brain barrier (BBB), regulation of the neurovascular system (e.g., vascular stability, vessel formation, cerebral blood flow, etc.), trafficking of inflammatory cells, clearance of toxic waste products from the brain, and acquisition of stem cell-like properties. In the neurovascular unit, pericytes perform these functions through coordinated crosstalk with neighboring cells including endothelial, glial, and neuronal cells. Dysfunction of pericytes contribute to a wide variety of diseases that lead to cognitive impairments such as cerebral small vessel disease (SVD), acute stroke, Alzheimer’s disease (AD), and other neurological disorders. For instance, in SVDs, pericyte degeneration leads to microvessel instability and demyelination while in stroke, pericyte constriction after ischemia causes a no-reflow phenomenon in brain capillaries. In AD, which shares some common risk factors with vascular dementia, reduction in pericyte coverage and subsequent microvascular impairments are observed in association with white matter attenuation and contribute to impaired cognition. Pericyte loss causes BBB-breakdown, which stagnates amyloid β clearance and the leakage of neurotoxic molecules into the brain parenchyma. In this review, we first summarize the characteristics of brain microvessel pericytes, and their roles in the central nervous system. Then, we focus on how dysfunctional pericytes contribute to the pathogenesis of vascular cognitive impairment including cerebral ‘small vessel’ and ‘large vessel’ diseases, as well as AD. Finally, we discuss therapeutic implications for these disorders by targeting pericytes.

Abstract 2746: Stat3 labels a subpopulation of reactive astrocytes required for brain metastasis

Abstract The diagnosis of brain metastasis involves high morbidity and mortality and remains as an unmet clinical need in spite of being the most common tumor in the brain. Brain metastasis affects between 10-30% of cancer patients with 200.000 new cases yearly only in the US. Increasing evidences point out the relevance of the microenvironment to understand the biology of brain colonization by metastatic cells, however translation strategies targeting it are lacking. We have identified altered signaling pathways in pro-metastatic reactive astrocytes (RA), and translated that into a novel therapeutic application for brain metastasis. Specifically we have identified a subpopulation of RA characterized by activated STAT3 pathway (pSTAT3+). This subpopulation is located in the vicinity of metastatic lesions intermingled with pSTAT3- RA in several experimental models and in 89% of human brain metastases independently of the primary tumor source. The pro-metastatic behaviour of RA is regulated by the activation of the STAT3 pathway, which modulates the immune system locally promoting the survival of cancer cells. Specifically the secretome of pSTAT3+ RA decreases the anti-tumor activity of CD8+ T cells as well as promotes the expansion of the pro-tumor population of CD74+ microglia/macrophage, which infiltrates metastasis cores. Interestingly, the immunosuppressive nature of pSTAT3+ RA is linked to the acquisition of stem cell-like properties, which might reflect the misuse of brain responses to injury instigated by the presence of cancer cells. Genetic and pharmacologic approaches targeting STAT3 in RA impair the progression of brain metastasis, even at advanced stages of the disease. Moreover, a safe and orally bioavailable STAT3 inhibitor reduced brain metastasis in 75% of 18 stage IV lung adenocarcinoma patients with established brain metastases, improving the outcome of the disease by increasing patient survival from 4 to 15 months. Besides increasing therapeutic opportunities for patients with brain metastasis, we have described for the first time the role of reactive astrocytes as regulators of local immunosuppression in brain metastasis as well as the importance of uncovering the heterogeneity within the metastasis-associated microenvironment. Citation Format: Neibla Priego, Lucía Zhu, Catia Monteiro, Manon Mulders, David Wasilewski, Wendy Bindeman, Laura Doglio, Elena Martínez-Saez, Santiago Ramón y Cajal, Coral Fustero-Torre, Elena Piñeiro-Yáñez, Aurelio Hernández-Laín, Valeria Poli, Javier A. Menéndez, Ricardo Soffietti, Joaquim Bosch-Barrera, Manuel Valiente. Stat3 labels a subpopulation of reactive astrocytes required for brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2746.

Abstract 3691: XPC inhibits lung cancer cell dedifferentiation by suppressing Snail expression

Abstract Lung cancer remains a leading cause of cancer-related deaths in the United States with unfavorable prognosis mainly due to tumor relapse and metastasis, which are recently believed to be caused by a specific population of cancer cells within tumor termed “cancer stem cells (CSCs)”. These cells share the common characteristic of self-renewal and differentiation as normal stem cell, but also show resistance to chemotherapy and radiation therapy. Thus, targeting CSC populations in lung tumors is critical to the prevention of tumor metastasis. Xeroderma pigmentosum group C (XPC) was first recognized as a DNA repair protein. As a DNA repair factor, XPC plays an important role in preventing carcinogenesis. However, it has also been reported that XPC insufficiency is associated with poor treatment outcomes for a variety of cancers, and low expression of XPC is correlated with poor prognosis of lung cancer patients, suggesting that XPC may suppress lung cancer progression. Here, we show that downregulation of XPC expanded lung CSCs characterized by CD133+, while overexpression of XPC limited this cell population, as well as reduced the tumorigenic potential of the lung cancer cell line. Furthermore, we found that XPC knockdown is able to promote the CD133--to-CD133+ cell conversion in A549 cells, indicating that XPC can inhibit lung cancer cell dedifferentiation. Mechanistic investigation demonstrated that XPC can suppress Snail expression by directly binding to the promoter region of the SNAI1 gene, leading to the enrichment of histone H3 trimethylation at serine 27 (H3K27me3) and loss of histone H3 acetylation at serine 27 (H3K27Ac) in this region. Given that Snail plays a critical role in the induction of epithelial-mesenchymal transition (EMT), which is a major mechanism for the acquisition of stem cell-like properties, we believe that XPC can limit the CSC population by inhibiting cancer cell dedifferentiation. In summary, we conclude that low expression of XPC in lung tumors can de-repress the expression of Snail, promote EMT, and increase the de novo production of CSCs, eventually facilitating lung tumor progression. Citation Format: Shurui Cai, Dayong Wu, Ananya Banerjee, Lu Liu, Chunhua Han, Tiantian Cui, Qi-En Wang. XPC inhibits lung cancer cell dedifferentiation by suppressing Snail expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3691.

Ectopic Expression of miR-147 Inhibits Stem Cell Marker and Epithelial-Mesenchymal Transition (EMT)-Related Protein Expression in Colon Cancer Cells.

Colon cancer is one of the most common cancers in the world. Epithelial-to-mesenchymal transition (EMT) is a crucial step in tumor progression and is also involved in the acquisition of stem cell-like properties. Some miRNAs have been shown to function as either tumor suppressors or oncogenes in colon cancer. Here we investigated the role of miR-147 in the regulation of the stem cell-like traits of colon cancer cells. We observed that miR-147 was downregulated in several colon cancer cell lines, and overexpressed miR-147 decreased the expression of cancer stem cell (CSC) markers OCT4, SOX2, and NANOG in the colon cancer cell lines HCT116 and SW480. Overexpressed miR-147 inhibited EMT by increasing the expression of epithelial markers E-cadherin and α-catenin while decreasing the expression of mesenchymal markers fibronectin and vimentin. Moreover, activation of EMT by TGF-β1 treatment significantly counteracted the inhibitive effect of miR-147 on the expression of CSC markers OCT4, SOX2, and NANOG, supporting the idea that overexpressing miR-147 inhibited stem cell-like traits by suppressing EMT in colon cancer. In addition, we found that overexpressed miR-147 downregulated the expression of β-catenin, c-myc, and survivin, which were related to the Wnt/β-catenin pathway. Moreover, treatment of miR-147 mimic-transfected cells with the Wnt/β-catenin pathway activator LiCl attenuated the inhibitive effect of the miR-147 mimic on the EMT and stem cell-like traits of colon cancer cells, indicating that ectopic expression of miR-147 inhibited stem cell-like traits in colon cancer cells by suppressing EMT via the Wnt/β-catenin pathway. In summary, our present study highlighted the crucial role of miR-147 in the inhibition of the stem cell-like traits of colon cancer cells and indicated that miR-147 could be a promising therapeutic target for colon cancer treatment.

Abstract 5506: NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties

Abstract Background: The NOTCH1 gene functions as either an oncogene or tumor suppressor in cancer depending upon the tumor type. Our group previously characterized the genomic alterations in head and neck squamous cell carcinoma (HNSCC), discovering that NOTCH1 is frequently altered with a pattern of inactivating mutations suggesting it is a tumor suppressor in this cancer type. However, recent work by others suggests NOTCH1 signaling plays a more complex role, possibly promoting a more aggressive phenotype or cancer stem cell-like properties in HNSCCs with wild type NOTCH1. Our present study aimed to systematically compare the phenotypic consequences of NOTCH1 signaling in HNSCC to better understand its function in cancer, and identify targets downstream of NOTCH1 signaling. Methods: Established HNSCC cell lines wild type for NOTCH1 (PJ34, FADU) or harboring an inactivating mutation (UMSCC22A) were engineered to express activated cleaved NOTCH1 (cl-NOTCH1) from a doxycycline-inducible promoter. In vitro cell growth was measured with clonogenic assays. Stem cell-like properties were measured by orosphere formation and anoikis resistance. Stem cell markers for HNSCC including Aldehyde dehydrogenase activity (ALDH), CD133, and CD44 expression were measured by flow cytometry. NOTCH1-regulated downstream gene expression changes were examined by RNA-seq and qRT-PCR. Results: Activation of NOTCH1 inhibited clonogenic growth of all three cell lines, regardless of original NOTCH1 gene status. Growth inhibition was frequently accompanied by spontaneous formation of spheroid-like structures, characteristic of stem cells. NOTCH1 activation in UMSCC22A and FADU cells promoted orosphere formation and anoikis resistance, conveying some stem cell-like properties. However, classical stem cell markers including ALDH activity, CD133, and CD44 expression were not affected by NOTCH1 activation. Furthermore, RNA-seq demonstrated that critical cancer-associated pathways, including proliferation, differentiation, and migration, were regulated by NOTCH1. NOTCH1 activation downregulated gene expression of ITGA3, ITGA4, ITGB1, ITGB6, and LAMC2, which are key adhesion molecules that human basal keratinocytes use for attachment to the basement membrane and maintenance of the stem cell compartment. Concomitantly, NOTCH1 activation increased the basal/superbasal marker SOX2, but also the early differentiation markers KRT4 and KRT13. SiRNA-mediated SOX2 silencing blocked NOTCH1-promoted anoikis resistance. Conclusion: NOTCH1 activation inhibits in vitro growth regardless of mutational status. We hypothesize that stem cell-like properties associated with NOTCH1 activation in HNSCC may be a consequence of pathways that recapitulate early differentiation, rather than true stem cell maintenance. Citation Format: Chenfei Huang, Shhyam Moorthy, Qiuli Li, Rami Saade, Jiping Wang, Xiayu Rao, Noriaki Tanaka, Jiexin Zhang, Lin Tang, Curtis R. Pickering, Patrick A. Zweidler-McKay, Abdullah A. Osman, Tong-Xin Xie, Eve Shinbrot, Liu Xi, David Wheeler, Adel K. El-Naggar, Jing Wang, Jeffrey N. Myers, Mitchell J. Frederick. NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5506.

PO-297 Oncogenic activity of SOX1 in gastric cancer

IntroductionGastric cancer (GC) remains one of the leading causes of global cancer mortality due to therapy resistance. The infection with Helicobacter pylori is the major risk factor, particularly in patients carrying H. pylori highly virulent strains. GC contains a sub-population of gastric cancer stem cells (gCSC) with capacity of self-renewal that are critical drivers of tumour initiation, recurrence and therapy resistance. SOX transcription factors play a key role in the regulation and maintenance of stem cells during the embryonic development and are also involved in cancer. SOX1 is a well-established tumour suppressor in several types of cancer and has an oncogenic role in glioblastoma, but its impact in GC remains largely unknown. Therefore, we aimed to elucidate its function in GC and its putative role in the action of H. pylori.Material and methodsFor this, we analysed GC patient samples, we co-cultured human GC cell lines with different H. pylori strains derived from patients of Donostia Hospital and we also performed functional studies of gain and loss of SOX1 function. Also, we performed transcriptomic analysis in SOX1-silenced cells and we carried out computational analysis using the ACRG datasets.Results and discussionsOur results revealed that SOX1 is highly expressed in human GC samples. Moreover, among a subset of SOX genes, SOX1 was the most significantly up-regulated in gCSC derived from GC cell lines and also in cisplatin resistant cells, as well as in response to H. pylori exposure, in a virulence-dependent manner. In GC cells, SOX1-silencing impaired self-renewal capacity in vitro and reduced tumorigenicity and tumour growth in vivo. The up-regulation of SOX1 showed the opposite phenotype, indicating that SOX1 exerts an oncogenic role in GC. Notably, we found that in GC cells SOX1 was required for H. pylori-induced proliferation, acquisition of stem cell-like properties and induction of β-catenin. Furthermore, the transcriptomic analysis revealed a significant alteration of the E2F signalling pathway in SOX1 knockdown cells. Consistently, E2F1 silencing phenocopied SOX1 knockdown. Moreover, the inhibition of SOX1 downregulated E2F1, suggesting that E2F1 could be a downstream effector of SOX1 in GC.ConclusionWe identify for the first time an oncogenic role of SOX1 in GC. Our findings establish that SOX1-E2F-b-catenin is a critical axis for gCSC maintenance and for H. pylori action, postulating that its inhibition could constitute a promising strategy to combat therapy resistance in GC.

MicroRNA-191, regulated by HIF-2α, is involved in EMT and acquisition of a stem cell-like phenotype in arsenite-transformed human liver epithelial cells

Inorganic arsenic is widely distributed in the environment, and epidemiologic data show a strong association between arsenic exposure and risk of liver cancer. An understanding of the mechanisms underlying development of liver cancer and metastasis would be useful in reducing the incidence and mortality of liver cancer. MicroRNAs (miRs) act as regulators in liver cancer. Here, we show that acute or chronic exposure of human liver epithelial L-02 cells to arsenite increased expression of miR-191. There were decreased levels of BASP-1 and E-cadherin and increased levels of WT-1 and N-cadherin, indicating that arsenite induced epithelial-mesenchymal transition (EMT). Moreover, arsenite increased EpCAM and CD90 mRNA levels, showing the acquisition of stem cell-like properties by these cells. Suppression of miR-191 resulted in repression of EMT and reduced expression of stem-cell markers. Further, a miR-191 inhibitor blocked spheroid formation and production of side population cells. Luciferase reporter assays indicated that miR-191 was a target of HIF-2α, and inhibition of miR-191 decreased the neoplastic and metastatic properties of arsenite-transformed L-02 cells. Thus, in arsenite-transformed liver epithelial cells, transcriptional activation of the miR-191 promoter by HIF-2α is involved in EMT and in the acquisition of a stem cell-like phenotype.

Abstract PR07: MicroRNA-130a regulates hematopoietic stem cell self-renewal and erythroid differentiation

Abstract Hematopoietic homeostasis is tightly regulated by controlling the balance between quiescence, self-renewal, and lineage-commitment of hematopoietic stem cells (HSCs). Deregulation of these processes and aberrant acquisition of stem cell-like properties is believed to be central to the pathogenesis of hematologic malignancies, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, little is known about the molecular networks maintaining the stem cell state and the epigenetic and post-transcriptional regulation of determinants that control these programs. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs. We have previously shown that miR-126 and miR-125a are differentially expressed across the human hematopoietic hierarchy and function to control self-renewal and cell fate decisions by reinforcing gene expression programs in a developmental stage-specific manner (Lechman et al. Cell Stem Cell, 2012; Wojtowitz et al. Cell Stem Cell, 2016). To identify additional miRNA(s) that play a functional role in hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were overexpressed (OE) in human CD34+CD38- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation. At 12 and 24 weeks after transplantation, enforced miR-130a expression (including an mOrange-mO+ indicator) conferred a competitive advantage over untransduced CB cells demonstrated by increased CD45+ human chimerism in the injected femur (IF), bone marrow (BM), and spleen of recipient mice. miR-130 enforced expression (miR-130a OE) increased the proportion of mO+/hCD45+ cells by approximately 2- and 5-fold after 12 and 24 weeks of repopulation, respectively. miR-130a OE xenografts showed multilineage engraftment with increased myeloid lineage output and significantly enhanced erythroid development at the expense of B-lymphoid lineage output in BM and spleen of recipient mice. Detailed flow cytometry analysis of xenografts revealed accumulation of immature GlyA+/CD71+/CD36+ erythroid progenitors, suggesting a differentiation block at the polychromic erythroblast stage. Notably, miR-130a OE induced the expansion of CD34+CD38- Lin- compartment and increased proportion of CD34+CD38-CD90+CD45RA- immuno-phenotypic HSC. Secondary transplantation involving limiting dilution analysis revealed approximately a 10-fold increase in HSC frequency, consistent with a role of miR-130a in HSC self-renewal. The lineage potential of miR-130OE primitive cells was assessed in vitro using single-cell stromal-based myelo-erythroid differentiation assay. Enforced expression of miR-130a in human HSC and multipotent progenitors (MPP) resulted in the decreased frequency of unipotent myeloid output (M colonies) and increased multipotent output (M/E/Meg, E/Meg colonies), supporting a role of miR-130a in erythroid-megakaryocytic fate specification. Label-free semiquantitative proteomics and subsequent gene set enrichment pathway analysis (GSEA) were performed on miR-130a OE and control transduced CD34+ CB cells to elucidate molecular mechanism(s) of miR-130a function. We identified that miR-130a modulated pathways centered on translational regulation and chromatin modification. Together, our data suggest that miR-130a plays a role in the regulation of the HSC self-renewal and erythroid differentiation. Given that several studies showed aberrant expression of miR-130a in MDS and some AML subtypes, it is important to delineate the role of miR-130a in normal hematopoiesis to comprehend its potential contribution to the development of hematologic malignancies. This abstract is also being presented as Poster 40. Citation Format: Gabriela Krivdova, Eric R. Lechman, Erwin M. Schoof, Veronique Voisin, Olga I. Gan, Aaron Trotman-Grant, Karin G. Hermans, Gary D. Bader, John E. Dick. MicroRNA-130a regulates hematopoietic stem cell self-renewal and erythroid differentiation [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR07.

Stem Cell-Like Properties of CK2β-down Regulated Mammary Cells.

The ubiquitous protein kinase CK2 has been demonstrated to be overexpressed in a number of human tumours. This enzyme is composed of two catalytic α or α’ subunits and a dimer of β regulatory subunits whose expression levels are probably implicated in CK2 regulation. Several recent papers reported that unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition, a process involved in cancer invasion and metastasis. Herein, through transcriptomic and miRNA analysis together with comparison of cellular properties between wild type and CK2β-knock-down MCF10A cells, we show that down-regulation of CK2β subunit in mammary epithelial cells induces the acquisition of stem cell-like properties associated with perturbed polarity, CD44high/CD24low antigenic phenotype and the ability to grow under anchorage-independent conditions. These data demonstrate that a CK2β level establishes a critical cell fate threshold in the control of epithelial cell plasticity. Thus, this regulatory subunit functions as a nodal protein to maintain an epithelial phenotype and its depletion drives breast cell stemness.

Abstract 3896: Exosomes derived from CD133+ hepatocellular carcinoma (HCC) cell promote the acquisition of stem cell-like properties of the targeted HCC cell via the Akt/GSK 3β/Snail and cyclin D1 pathways

Abstract Backgrounds and Aims: Liver cancer stem cells are closely related to the recurrence and metastasis of hepatocellular carcinoma (HCC). However, the mechanism underlying the reciprocity between liver cancer stem cells and HCC cells in the tumor microenvironment remains unclear. Exosomes act as natural shuttles to deliver bioactive molecules that alter the behavior of the recipient cells, offering a new means of cell-cell communication for physiological and pathological processes in the liver. This study aimed to clarify whether the exosomes derived from the liver stem cells could facilitate the acquisition of stem cell-like properties of the adjacent HCC cells, thereby promoting the malignant process of HCC. Methods: CD133+ liver cancer stem cells were isolated from the highly invasive HCC-LM3 cells or tumor tissues of HCC patients using flow cytometry and were identified using qPCR and immunofluorescence. The exosomes from CD133+ liver cancer stem cells were identified using CD133 and other stem cell markers, and they were then used to treat HepG2 and Hep3B cells. The cell viability, invasion ability, sphere formation and tumorigenesis abilities of the treated HepG2 and Hep3B cells were analyzed to reveal the effect of CD133+ exosomes, and the underlying molecular mechanism was further explored. Results: CD133+ liver cancer stem cells were successfully isolated from HCC-LM3 cells and HCC patients’ tumor tissues, which were able to release CD133+ exosomes. The intake of CD133+ exosomes promoted the putative stem cell phenotype of Hep3B and HepG2 cells, as evidenced by significantly enhanced cell proliferation, invasion and sphere formation abilities in vitro and tumorigenesis in vivo. In additional, we found that CD133+ exosomes activated the PI3K/Akt/GSK3β/Snail and cyclin D1 pathway, thereby reducing the expression of E-Cadherin and promoting the epithelial-mesenchymal transition (EMT) of HCC cells. Conclusions: Our results suggest that exosomes derived from CD133+ HCC cells can change the malignant behavior and promote the acquisition of stem cell-like properties of targeted HCC cells via the Akt/GSK 3β/Snail and cyclin D1 pathways. Note: This abstract was not presented at the meeting. Citation Format: Pengyi Guo, Haitao Yu, Xiaozhai Xie, Yi Wang, Gang Chen. Exosomes derived from CD133+ hepatocellular carcinoma (HCC) cell promote the acquisition of stem cell-like properties of the targeted HCC cell via the Akt/GSK 3β/Snail and cyclin D1 pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2017-3896

Acquisition of anticancer drug resistance is partially associated with cancer stemness in human colon cancer cells.

Colorectal cancer (CRC) is one of the most aggressive cancers worldwide. Several anticancer agents are available to treat CRC, but eventually cancer relapse occurs. One major cause of chemotherapy failure is the emergence of drug-resistant tumor cells, suspected to originate from the stem cell compartment. The aim of this study was to ask whether drug resistance was associated with the acquisition of stem cell-like properties. We isolated drug-resistant derivatives of two human CRC cell lines, HT29 and HCT116, using two anticancer drugs with distinct modes of action, oxaliplatin and docetaxel. HT29 cells resistant to oxaliplatin and both HT29 and HCT116 cells resistant to docetaxel were characterized for their expression of genes potentially involved in drug resistance, cell growth and cell division, and by surveying stem cell-like phenotypic traits, including marker genes, the ability to repair cell-wound and to form colonospheres. Among the genes involved in platinum or taxane resistance (MDR1, ABCG2, MRP2 or ATP7B), MDR1 was uniquely overexpressed in all the resistant cells. An increase in the cyclin-dependent kinase inhibitor p21, in cyclinD1 and in CD26, CD166 cancer stem cell markers, was noted in the resistant cells, together with a higher ability to form larger and more abundant colonospheres. However, many phenotypic traits were selectively altered in either HT29- or in HCT116-resistant cells. Expression of EPHB2, ITGβ-1 or Myc was specifically increased in the HT29-resistant cells, whereas only HCT116-resistant cells efficiently repaired cell- wounds. Taken together, our results show that human CRC cells selected for their resistance to anticancer drugs displayed a few stem cell characteristics, a small fraction of which was shared between cell lines. The occurrence of marked phenotypic differences between HT29- and HCT116-drug resistant cells indicates that the acquired resistance depends mostly on the parental cell characteristics, rather than on the drug type used.

Abstract P1-16-06: Luteolin inhibits progestin-dependent VEGF induction, stem-cell like characteristics, and tumor progression of human breast cancer cells

Abstract Clinical trials and epidemiological evidence show that combined estrogen (E) and progestin (P) hormone replacement therapy (HRT) increases the risk of breast cancer in postmenopausal women, whereas HRT containing E alone does not. Tumor progression is dependent on angiogenesis, which provides nutrients vital to the developing cancer. We previously showed, both in vitro and in vivo, that natural and synthetic P (including the widely used progestin Medroxyprogesterone acetate, MPA), increase production of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in human breast cancer cells (Cancer Res., 1998, 58:392). This effect is blocked by the anti-progestin RU-486, suggesting involvement of progesterone receptors in the process (Int J Cancer, 2001, 92:469). Evidence from our laboratory using in vivo breast cancer models suggests that P accelerates the development of tumors from latent tumorigenic cells. This leads to the formation of palpable tumors and tumor metastasis, processes that may be attributed to increased production of VEGF (Cancer Res., 2007, 67:9929; Menopause, 2010, 17:1040). RU-486 blocks P-dependent VEGF production and thereby reduces tumor growth; however, the anti-progestin has severe side-effects averting its long-term use. Recently, we have studied less toxic naturally-occurring compounds for their ability to antagonize P-dependent VEGF induction and block tumor progression. In this study, we tested the effects of luteolin, a flavonoid commonly found in fruits and vegetables, on proliferation of BT-474 and T47-D breast cancer cells and their P-dependent production of VEGF. Luteolin treatment (25-100 μM) for 24-48 h reduced in vitro tumor cell viability and induced apoptosis. Interestingly, treatment with a lower concentration of luteolin (10 μM) blocked the production of P-dependent VEGF, indicating that VEGF suppression precedes luteolin-mediated loss of cell viability. Furthermore, luteolin (20 mg/kg, i.p.) suppressed the growth of MPA-dependent T47-D human xenograft tumors in nude mice. Immunohistochemical analysis showed that luteolin reduced P-induced VEGF in tumor sections (p<0.05). These findings strongly suggest that the flavonoid disrupts tumor progression by blocking P-dependent angiogenesis and preventing tumor cell proliferation. Furthermore, luteolin blocked the MPA-induced acquisition of stem cell like properties by breast cancer cells; CD44 expression, ALDH activity and mammosphere formation were all reduced by the flavonoid. We contend therefore that luteolin is a compound with valuable therapeutic properties. Its ability to reduce levels of VEGF, coupled with its capacity to interfere with the acquisition of stem-cell like properties by breast cancer cells, make luteolin a compound with significant clinical potential in the battle against P-dependent human breast cancer. Supported by a COR award from the College of Veterinary Medicine and in part by funds from generous donors to the Ellis Fischel Cancer Center, University of Missouri. Citation Format: Hyder SM, Cook MT, Besch-Williford C, Liang Y. Luteolin inhibits progestin-dependent VEGF induction, stem-cell like characteristics, and tumor progression of human breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-16-06.

Abstract B35: Concurrent use of alisertib and vaccinia virus demonstrate increased in vitro efficacy against breast cancer models with elevated numbers of cancer stem-like cells

Abstract Background: Accumulating evidence indicates that breast cancer progression, resistance to therapies and development of metastasis are driven by cancer cells with stem cell-like characteristics. The increased numbers of cancer stem-like cells (CSCs) have been reported in triple negative breast cancers (TNBCs). Therefore, novel methods for targeting CSC population are needed. Aurora-A kinase (AURKA) has been shown to play a key role in acquisition of stem cell-like properties by breast cancer cells. Furthermore, the CSCs have been demonstrated to have increased sensitivity to AURKA specific inhibitor, MLN8237 (Alisertib). Our group has previously reported that oncolytic vaccinia virus (OVV) preferentially infects CSCs. Therefore we hypothesize that the combined treatment of OVV and AURKA inhibitor will result in overall enhanced anti-tumor activity due to improved elimination of CSCs. Results: We confirmed that AURKA overexpression in MCF-7 breast cancer cell line (MCF-7 AURKA) resulted in increased number of CSCs as shown by upregulation of CD44 antigen and decreases in expression of CD24 antigen. The MCF-7 and MCF-7 AURKA cells were subsequently infected with 0.1 pfu/cell of OVV expressing EGFP (OVV-EGFP). After 72h of incubation, viral replication was analyzed by fluorescent microscopy and standard plaque-forming assay. The analysis revealed over 2-fold increases in viral replication in MCF-7 AURKA cells as compared to the parental counterparts. The enhanced viral replication resulted in decreased viability of MCF-7 AURKA cells when compared to OVV-treated MCF-7 (64% vs 81%, p<0.031). The efficacy of combined treatment of OVV-EGFP and alisertib was next analyzed in MCF-7AURKA cells. The MTT assay analysis revealed 73% decreases in viability of cells treated with combination of oncolytic viortherapy and alisertib, whereas the effect of single treatment resulted in 34% and 52% reduction of viability for OVV-EGFP and alisertib, respectively (p <0.01 and p< 0.024). We next tested the efficiency of OVV-EGFP and Alisertib combination treatment against TNBC cell line, BT549. The cells treated with combination of OVV-EGFP and alisertib showed 63% decreases in cell viability compared to 25% and 46% reductions mediated by OVV-EGFP and alisertib, respectively (p<0.003 and p< 0.03). Conclusions: Combination therapy of alisertib and OVV has a significantly higher anti-cancer cell activity in vitro than either agent used alone, and it is particularly applicable for treatment of cells with CSC phenotype. In vivo studies are currently in progress. The combined treatment of alisertib and OVV represents novel therapeutic approach in treating breast cancer with increased numbers of CSCs, resistance to standard therapies and worse clinical outcomes. The results present a novel treatment approach with high translational potential. Citation Format: Malgorzata Gil, Marcin Komorowski, Danuta Kozbor, Mateusz Opyrchal. Concurrent use of alisertib and vaccinia virus demonstrate increased in vitro efficacy against breast cancer models with elevated numbers of cancer stem-like cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B35.

Stemness-related transcriptional factors and homing gene expression profiles in hepatic differentiation and cancer.

Stem cell transcriptional signature activation is an essential event in the development of cancer. This study aimed to investigate the differential expression profile of three pluripotency-associated genes (OCT4, NANOG, and SOX2), G-protein-coupled chemokine receptor 4 (CXCR4) and the ligand (CXCL2), and alpha feto-protein (AFP) in hepatogenic differentiated stem cells and in sera of hepatitis C virus (HCV) and HCV-induced hepatocellular carcinoma (HCC) patients. Mesenchymal stem cells derived from umbilical cord blood were differentiated using hepatogenic differentiation media. Serum specimens were collected from 96 patients (32 cirrhotic HCV, 32 early HCC, and 32 late HCC) and 96 controls. Real-time quantitative reverse transcription polymerase chain reaction was performed for relative quantification of the 6 target genes using LIVAC method. In silico network analysis was also executed to explore the pluripotency and tumorigenic regulatory circuits in liver cancer. The expression levels of all genes declined gradually during the stages of stem cell differentiation. On univariate and multivariate analyses, NANOG, CXCR4 and AFP were significantly up-regulated in HCC patients with late clinical stage. In contrast, SOX2 and CXCL2 were markedly over-expressed in cirrhotic patients and could be used for clear demarcation between cirrhotic and HCC patients in our cases. In conclusion, our data highlight the potential role of SOX2 stem cell marker and CXCL2 chemokine in liver cell degeneration and fibrogenesis in HCV-induced hepatic cirrhosis in our sample of the Egyptian population. In addition, the significant association of NANOG and CXCR4 high-expression with late HCC, could contribute to the acquisition of stem cell-like properties in hepatic cancer and dissemination in late stages, respectively. Taken together, our results could have a potential application in HCC prognosis and treatment.

Abstract LB-B02: Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells

Abstract [Background] Most NSCLC patients harboring activating EGFR mutations benefit from treatment with EGFR-TKIs, but the final clinical efficacy of EGFR-TKIs varies because of development of tumor resistant to EGFR-TKIs. Multiple kinase-targeted 2nd generation TKIs such as afatinib have been developed to overcome drug resistance to the 1st generation TKIs. Afatinib is an irreversible multitargeted TKI targeting EGFR including T790M, HER2 and HER4. To develop further personalized therapeutics and drug resistance modifiers, we should understand the molecular based mechanism of drug resistance to 2nd as well as 3rd generation TKIs. In our present study, we present a novel finding that acquisition of cancer stem-like cells properties accompanying with activation of residual Src family kinase (SFK)/focal adhesion kinase (FAK) is responsible for the survival of afatinib-resistant lung cancer cells when expression of targeted EGFR, HER2 and HER4 is abraded. [Materials and methods] We have established afatinib-resistant subclones from human lung cancer cell lines, HCC827, harboring activating EGFR mutation after stepwise exposure to afatinib. The afatinib-resistant clones do not harbor T790M, K-ras mutation or PTEN loss. We have characterized biochemical properties of drug-resistant subclones as compared with their drug-sensitive counterparts by Western blot and RT-PCR. ALDEFLUOR kit was used to isolate cell populations with the stem cell specific ALDH enzymatic activity. [Results] We characterized two independent afatinib-resistant subclones, BR1-8 and BR2-3. [1] Afatinib-resistant subclones showed markedly decreased expression of EGFR, HER2, HER3, c-Met and PDGFRβ as compared with their parental cell lines, HCC827; [2] Akt phosphorylation in afatinib-resistant subclones was not suppressed by afatinib; [3] SFK and FAK were activated in resistant subclones, and combination of afatinib with c-Src siRNA or with dasatinib moderately suppressed cell growth and AKT phosphorylation; [4] Combination of afatinib with siFAK moderately blocked cell growth of resistant subclones. [5] Cancer stem like cell marker, ALDH1, positive cell population increased in afatinib-resistant sublines compared with HCC827 cell lines. [Conclusions] Together, our present study presents that increase of cancer stem like cells and SFK/FAK activation could be a mechanism responsible for acquired resistance to afatinib. We would further discuss the association of ALDH1 expression and SFK/FAK activation with afatinib resistance in lung cancer. Citation Format: Yuichi Murakami, Kahori Sonoda, Koichi Azuma, Kosuke Watari, Michihiko Kuwano, Mayumi Ono. Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B02.

Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells.

Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance.