Stem Cell-like Characteristics Research Articles

The Notch Pathway Promotes Osteosarcoma Progression through Activation of Ephrin Reverse Signaling.

Despite significant advancements in the diagnosis and treatment of osteosarcoma, the molecular mechanisms underpinning disease progression remain unclear. This work presents strong clinical and experimental evidence demonstrating that Notch signaling contributes to osteosarcoma progression. First, using a cohort of 12 patients, Notch genes were upregulated in tumors compared with adjacent normal tissue, and high tumor expression of Notch1 intercellular domain (NICD1) and the Notch target gene Hes1 correlated with poor chemotherapy response. Data mining of publicly available datasets confirmed that expression of Notch pathway genes is related to poor prognosis in osteosarcoma. On the basis of in vitro analysis, Notch signaling promoted osteosarcoma proliferation, enhanced chemoresistance, facilitated both migration and invasion, and upregulated stem cell-like characteristics. Xenograft models demonstrated that Notch signaling promotes primary tumor growth and pulmonary metastasis, and Notch inhibition is effective in reducing tumor size and preventing metastasis. Mechanistically, activated Notch signaling induces the expression of ephrinB1 and enhances the tumor-promoting ephrin reverse signaling. Overall, these findings provide functional evidence for Notch pathway genes as candidate biomarkers to predict prognosis in patients with osteosarcoma, and suggest a mechanistic rationale for the use of Notch inhibitors to treat osteosarcoma. IMPLICATIONS: The study provides preclinical evidence for Notch pathway as a molecular marker to predict osteosarcoma prognosis and as a therapeutic target against osteosarcoma. In addition, we identified a novel mechanism that ephrin reverse signaling acts as a key mediator of Notch pathway.

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

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.

Ing4 Regulates Stress Hematopoiesis and Represses Self-Renewal in Multipotent Progenitor Cells

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells and is a frequent target of inactivation in various cancer types. The tumor suppressive activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In zebrafish, loss of Ing4 results in loss of HSC specification and a significant increase in NF-kB target gene expression. Knockdown of NF-kB expression in Ing4 deficient zebrafish recovered HSC marker expression in the aorta suggesting that NF-kB inhibition could remediate the loss of Ing4 expression. Small molecule NF-kB pathway inhibitors with varying mechanisms were also observed to rescue of HSC marker staining in the zebrafish aorta. Ing4 deficient embryos incubated with a lower dose of inhibitor had a 31% recovery of marker staining and 82% of embryos incubated in the highest dose recovered HSC marker staining emphasizing a dose dependent rescue of HSC specification through NF-kB suppression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- bone marrow has aberrant hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with a concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Analysis of differentiation in Ing4 null bone marrow also reveals skewed hematopoiesis. We see a 14% increase in granulocytes in the null mouse marrow and observe similar skewing in CFU assays. Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. In further characterization of these cells, we found an increase in MPPs that express lower levels of CD34 (55.5% vs 67.7%). CD34 expression is a marker of HSCs. This CD34+/mid population also express CD229 (85% positive), which is barely detectable in wildtype marrow (less that 0.01%). CD229 is also an HSC marker. Based on these exciting findings, we hypothesize that we have identified a subset of CD34+/midCD229+ MPPs in Ing4 deficient mice that retain self-renewal characteristics. Our data suggest that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures Zon: CAMP4: Equity Ownership; Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership.

Circulating Endometrial Cells: A New Source of Information on Endometriosis Dynamics.

The focus of the presented work was to isolate and characterize circulating endometrial cells (CECs) enriched from peripheral blood (PB) of patients with diagnosed endometriosis. The molecular characteristics of CECs could be supportive for an understanding of endometriosis pathogenesis and treatment decisions in the future. Material and Methods: Blood samples (n = 423) were tested for CECs presence. Subsequently, gene expression analysis (GEA) was carried out for CECs. In parallel, CECs presence and characteristics were tested during menstrual cycle (MC) phases in 11 patients. CECs were enriched by size-based separation. Results: CECs were present in 78.4% of the tested blood samples. In line with the revised American Fertility Society (rAFS) classification, CECs presence was confirmed in all the acknowledged endometriosis stages: minimal, mild, moderate, and severe. Surprisingly, CECs negativity rate was also reported for severe disease in 21.1% of cases. The CECs captured during MC phases displayed different cytomorphology, including epithelial, stromal, and stem cell-like characteristics. The highest CECs numbers were detected in the mid-secretory phase of MC, which corresponds to uterine lining decidualization. CECs captured during mid-secretory periods expressed genes KRT18, NANOG, and VIM in higher amounts when compared to the proliferative phase of MC, where genes KRT19 and ESR1 were mostly elevated. GEA of the super-positive CECs samples (1000 CECs/8 mL PB) revealed high expression of genes KRT18, VIM, NANOG, and FLT1. The expression of these genes was also elevated in the endometriosis tissue samples and endometrioma. Conclusion: The panel of the identified CEC genes could be tested in a prospective manner to confirm the role of CECs in endometriosis pathogenesis and diagnostics.

CSIG-12. PREX1 LINKS ABERRANT PI 3-KINASE PATHWAY SIGNALING TO MAINTENANCE OF THE NEURAL STEM CELL-LIKE PHENOTYPE OF GLIOBLASTOMA CELLS

Abstract Virtually all primary glioblastomas have aberrant activation of the PI 3-kinase pathway through mutational inactivation of tumour suppressors such as PTEN and/or mutational activation of oncogenes. Most glioblastomas also overexpress the Rac guanine nucleotide exchange factor PREX1, which is activated by PI 3-kinase pathway signaling. To determine the role of PREX1 in mediating the effects of aberrant PI 3-kinase pathway signaling in glioblastoma, we used CRISPR/Cas9 to knockout PREX1 in patient-derived glioblastoma cells cultured under conditions that maintain their neural stem cell-like characteristics. Multiple PREX1-null glioblastoma cell clones showed reduced motility and an altered morphology, with a smaller cell body and multiple neurite-like extensions. They also had increased expression of doublecortin, a marker of committed neurogenic progenitor cells. Analysis of RNA-seq data for transcription factor usage was consistent with the acquisition of an intermediate progenitor-like phenotype in PREX1-null cells, with loss of neural stem cell-associated polycomb repressive complex 2 and DMRTA2 activity and the expression of an EOMES/TBR2 signature characteristic of intermediate progenitors. The reduced motility, altered morphology and increase in doublecortin protein levels could all be reversed by re-expression of PREX1. PREX1-null cells had a reduced ability to phosphorylate and inactivate Lgl1, which is required for the maintenance of neural stem cell-like glioblastoma cell populations in vivo. These data show that PREX1 links aberrant PI 3-kinase signaling to maintenance of the neural stem cell-like phenotype in glioblastoma cells.

Melatonin decreases in vitro viability and migration of spheres derived from CF41.Mg canine mammary carcinoma cells

BackgroundMammary cancer is a common disease affecting female dogs, where approximately 50% of the cases are malignant. There is a subpopulation of cancer cells with stem cell-like features within the tumour microenvironment, which can form in vitro spheres, cell structures that grow in anchor-free conditions. This cell population shows resistance to conventional antitumor treatments explaining in part the recurrence of some type of cancers. It has been previously reported that spheres derived from CF41.Mg canine mammary carcinoma cells exhibit several stemness features. Melatonin has shown antitumor effects on cancer mammary cells; nevertheless, its effects have been poorly evaluated on canine mammary cancer stem-like cells. In this regard, it has described that melatonin decreases the expression of OCT-4 in CMT-U2229 mammary cancer cells, a transcription factor that participates in the modulation of self-renewal and drug resistance in cancer stem-like cells. The aim of this study was to compare the effects of melatonin on viability and migration of canine mammary carcinoma CF41.Mg-spheres, and CF41.Mg-parental cells. CF41.Mg cells were grown in DMEM high-glucose medium containing 10% bovine foetal serum. CF41.Mg-spheres were cultured in ultra-low attachment plates with serum-free DMEM/F12 containing several growth factors. Cell viability (MTS reduction) and migration (transwell) assays were conducted in presence of melatonin (0.01, 0.1 or 1 mM).ResultsMelatonin decreased cell viability at 1 mM (P < 0.05), with a significant reduction in spheres compared to parental cells at 24 and 48 h (P < 0.05). Cell migration was inhibited in response to non-cytotoxic concentration of melatonin (0.1 mM) (P < 0.05) in spheres and monolayer of cells, no significant differences were detected between both cell subtypes.ConclusionsThese results indicate that melatonin reduces viability and migration of CF41.Mg cells, where spheres exhibit greater sensitivity to the hormone. Thus, melatonin represents a valuable potential agent against mammary cancer cells, especially cancer stem-like cells.

Calcitonin induces stem cell-like phenotype in prostate cancer cells.

Stem cell-like-cancer cells are key drivers of tumor growth, metastasis, and relapse of cancer following remission. Prostate stem cell-like cancer cells isolated from human prostate cancer (PC) biopsies express CD44+/α2β1 hi/CD133+ cell surface markers and can self-renew in vitro. Expression of calcitonin (CT) and its receptor (CTR) is frequently elevated in PCs and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We investigated whether CT-CTR autocrine axis induces stem cell-like phenotype in two PC cell lines. CT-CTR axis in these cell lines was activated by enforced expression of CTR. The cells were then examined for the changes in the expression of CD44 and CD133, collagen adherence, tumorigenic, metastatic and repopulating characteristics. The activation of CT-CTR axis led to a large increase in adherence to collagen and a remarkable increase of CD44 and CD133 in PC-3 and LNCaP cells. This was accompanied by a strong increase in tumorigenic, metastatic and repopulation properties of PC cells. However, the mutation of CTR-C PDZ-binding site in CTR almost abolished CTR-mediated increases in stem cell-like characteristics of PC cells. These results support an important role for CT-CTR axis in the progression of PC from localized cancer to an aggressive form, and a majority of proinvasive CTR actions may be mediated through its interaction with its partner protein at the PDZ-binding site. These results suggest that CT/CTR can serve as a valuable target to prevent the generation of stem-like PC cells.

Evaluation of the cancer stem cell marker DCLK1 in patients with lymph node metastases of head and neck cancer

BackgroundLymph node metastases are frequently detected in head and neck squamous cell carcinoma (HNSCC) patients. Little is known about biomarkers expressed in lymph node metastases or their influence on clinical outcome. Doublecortin-like kinase 1 (DCLK1) is one marker that might be associated with outcome, owing to its correlation with stem cell-like characteristics. MethodsWe assessed the expression of DCLK1 in 74 postoperatively irradiated patients in histologically confirmed HNSCC lymph node metastases. Statistical analysis of the association with DCLK1 on clinical outcomes was performed. ResultsDCLK1 was expressed in 63.5% of our patient cohort. DCLK1(+) HNSCC patients, compared with those without DCLK1 expression, showed a significantly poorer time to recurrence. Moreover, we observed a significantly poorer time to recurrence in HPV(-) HNSCC patients, and significantly shorter overall and disease-free survival rates in HPV(-) oropharyngeal cancer patients, compared with HPV(+) patients with these cancers. HPV(+) patients showed no significant differences in survival time according to DCLK1 expression. However, recurrent disease occurred in only DCLK1(+) patients. Mulitivariate analysis showed that DCLK1 expression in lymph node metastases is an independent marker for recurrence. ConclusionDCLK1 expression might be associated with poorer clinical outcomes in HNSCC patients, specifically in HPV(-) move patients. However, larger studies are required to verify our results.

Tex10 promotes stemness and EMT phenotypes in esophageal squamous cell carcinoma via the Wnt/β‑catenin pathway.

A previous study by our group suggested that testis expressed 10 (Tex10) contributes to tumor progression by promoting stem cell-like features in hepatocellular carcinoma. However, the relevance of pluripotency factor Tex10 in esophageal squamous cell carcinoma (ESCC) has remained elusive. The objective of the present study was to investigate the role of Tex10 in ESCC. For this purpose, the mRNA and protein expression of Tex10 was detected by reverse transcription-quantitative PCR, western blot analysis and immunohistochemistry. In a loss-of-function experiment, EC109 cells were transfected with lentiviral vectors containing Tex10 short hairpin RNA or negative control. Cell proliferation was assessed using a Cell Counting kit-8, and flow cytometry was used to analyze apoptosis and the cell cycle. Transwell assays were employed to examine the migratory and invasive capacity, and a sphere formation assay was performed to assess the clonogenicity of the EC109 cells. The results revealed that the elevated expression of Tex10 was positively associated with malignancy and with epithelial-mesenchymal transition (EMT)-associated mesenchymal markers in human ESCC specimens. The knockdown of Tex10 led to the inhibition of cell proliferation, the induction of apoptosis and cell cycle arrest, and decreased the stemness, migratory and invasive capacity of the EC109 cells. Furthermore, the silencing of Tex10 enhanced the sensitivity of the ESCC cells to 5-fluorouracil. In addition, the present study revealed that Tex10 plays an essential role in regulating EMT via the activation of Wnt/β-catenin signaling. On the whole, the findings of the present study suggest that the downregulation of Tex10 in ESCC specimens is significantly associated with tumor malignancy, and that Tex10 promotes stem cell-like features and induces the EMT of ESCC cells through the enhancement of Wnt/β-catenin signaling.

Glioblastoma initiating cells are sensitive to histone demethylase inhibition due to epigenetic deregulation.

Tumor-initiating cells are a subpopulation of cells that have self-renewal capacity to regenerate a tumor. Here, we identify stem cell-like chromatin features in human glioblastoma initiating cells (GICs) and link them to a loss of the repressive histone H3 lysine 9 trimethylation (H3K9me3) mark. Increasing H3K9me3 levels by histone demethylase inhibition led to cell death in GICs but not in their differentiated counterparts. The induction of apoptosis was accompanied by a loss of the activating H3 lysine 9 acetylation (H3K9ac) modification and accumulation of DNA damage and downregulation of DNA damage response genes. Upon knockdown of histone demethylases, KDM4C and KDM7A both differentiation and DNA damage were induced. Thus, the H3K9me3-H3K9ac equilibrium is crucial for GIC viability and represents a chromatin feature that can be exploited to specifically target this tumor subpopulation.

Primary colonospheres maintain stem cell-like key features after cryopreservation.

The development of efficient and repeatable protocols for biobanking and prolonged storage of cancer stem cells (CSCs), with minimum alterations in biological function, is valuable and desired, particularly for retrospective analysis and clinical applications. In particular, data regarding the effect of cryopreservation on CSCs's functional features is scarce. In this regard, few studies have been shown that 3D spheroid structures, which enriched for CSCs, can keep their biological phenotype and genetic profiles. Here, for the first time, we present data on cryopreservation of CT-26 colonospheres, with the focus on essential stem cell-like properties after thawing. Tumor biopsy-derived colonospheres were frozen in standard freezing media (90% fetal bovine serum + 10% dimethyl sulfoxide) and stored in liquid nitrogen for 10 months. Then, cryopreservation effect on preservation of CSCs-related features was verified using real-time polymerase chain reaction for evaluation of stemness genes and flow cytometry for the putative colorectal CSC surface biomarkers. The self-renewal capacity of thawed spheres was also compared with their fresh counterparts using serial formation assay. Finally, tumorigenic capacity of both groups was evaluated in immunocompetence mouse model. Our data indicated that postthawed colonospheres had high viability without drastic alteration in biological and structural features and maintained self-renewal potential after sequential passages. Real-time analysis showed that both fresh and frozen colonospheres displayed similar expression pattern for key stemness genes: SOX2 and OCT4. Cryopreserved spheroids expressed CD133, CD166, and DCLK1 CSCs surface biomarkers at elevated levels when compared with parental as non-cryopreserved counterparts. Our electron scanning microscopy micrographs clearly demonstrated that postthawed colonospheres retain their integrity and cell surface morphology and characteristics. We also found that both fresh and frozen spheroids were equally tumorigenic. This study represented an effective strategy for reliable storage of intact CT-26 colonospheres; this can provide researchers with a functionally reliable repository of murine colorectal CSCs for their future CSCs projects.

Long non-coding RNA MEG3 regulates migration and invasion of lung cancer stem cells via miR-650/SLC34A2 axis

Long non-coding RNA maternally expressed gene 3 (MEG3) is related to the occurrence and development of non-small cell lung cancer (NSCLC). However, the function and underlying molecular mechanisms of MEG3 in lung cancer stem cells (LCSCs) are still unclear. LCSCs were determined in lung cancer cells using fluorescence-activated cell sorting (FACS). qRT-PCR and western blot were performed to examine the expressions of MEG3, miR-650, solute carrier family 34 member 2 (SLC34A2), octamer-binding transcription factor 4 (Oct4), and CD133. Sphere assay was employed to evaluate sphere-forming ability. Cell migration and invasion were analyzed by Transwell assay. The relationships among MEG3, miR-650, and SLC34A2 were validated by luciferase reporter, RIP, and RNA pulldown assays. We found MEG3 was downregulated in LCSCs. MEG3 depletion strengthened stem cell-like characteristics and sphere-forming ability in LCCs. Upregulation of MEG3 suppressed migration and invasion in LCCs and LCSCs. miR-650 was bound to MEG3 and upregulated in LCSCs. miR-650 inhibitor alleviated si-MEG3-induced promotion of stem cell-like characteristics in lung cancer cells (LCCs) H1299. Furthermore, miR-650 mimic attenuated the MEG3 upregulation-mediated inhibition of migration and invasion. In addition, SLC34A2 was a target of miR-650 and downregulated in LCSCs. miR-650 mimic induced stem cell-like characteristics in LCCs, which was weakened by overexpression of SLC34A2. In contrast, the repression of SLC34A2 mitigated the miR-650 silencing-induced inhibition of migration and invasion in LCCs and LCSCs. Besides, MEG3 regulated SLC34A2 expression by sponging miR-650. Importantly, SLC34A2 weakened MEG3-mediated stem cell-like state and cell metastasis. Our data suggested MEG3 was involved in stem cell-like state of LCCs and curbed migration and invasion through miR-650/SLC34A2 axis in NSCLC.

319P - The role of AXL as mechanism of resistance to trastuzumab and a prognostic factor in breast cancer HER2 positive: A translational approach

BackgroundBreast cancer (BC) is a heterogeneous disease. HER2+ BC represents between 15-30% of cases. Trastuzumab (T), a monoclonal antibody, has been successfully improved clinical benefits in both adjuvant and in metastatic settings. Despite this evidence, many patients experience resistance to therapy. The objective of this study is to assess AXL as a potential mechanism of resistance and its implication as a prognostic factor. MethodsWe used three cell lines with acquired resistance to T. Resistant models were generated by treating parental cells (AU565, SKR3, BT474) with constant dose of T (15mg/mL) for 6 months. Cell viability was estimated by MTT assay. Proteins were assessed by Western blot (WB) and flow cytometry and genes by qRT-PCR. AXL was downregulated by siRNA and a selective AXL inhibitor (TP-0903). The prognostic value of AXL was evaluated in primary tumor in a cohort of HER2+ BC patients treated with T in adjuvant setting from Hospital Clínico València (n=33). ResultsAcquired resistant cell lines (RCL) maintained HER2 overexpression. Cells were more proliferative and presented an increase in stem cell-like characteristics compared to sensitive parental cell lines. There was an important up-regulation of AXL (>2.5 fold-change) and epithelial-mesenchymal transition markers (VIM, CDH2, and FN1) in RCL (p<0.05). Sensibility to T was restored by silencing AXL and with TP-0903 treatment decreasing cell viability and IC50 of T (p<0.05). AXL expression was associated with metastasis in a cohort of HER2+ BC patients (p<0.001). There was no difference in GAS6 (a ligand of AXL). The role of AXL was also evaluated in a public data set and it was related with worse prognosis (p<0.001). ConclusionsOur results suggest: 1) RCL were more proliferative, more mesenchymal-like and stem cell-like properties; 2) AXL was a potential mechanism of secondary resistance to T; 3) Combination therapy with AXL inhibitor plus T restored sensitivity in in vitro model with AXL overexpression; 4) AXL expression was associated with relapse in HER2+ BC patients. These results showed AXL as a prognostic factor and a potential therapeutic target in HER2+ patients with resistance to T. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureA. Lluch: Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy: Eisai; Advisory / Consultancy: Celgene. All other authors have declared no conflicts of interest.

MiR-133b suppresses colorectal cancer cell stemness and chemoresistance by targeting methyltransferase DOT1L

Cancer stem cells (CSCs) are a subpopulation of chemoresistant cells that play a critical role in disease recurrence following chemotherapy. It has been reported that microRNA-133b (miR-133b) acts as a tumor suppressor in colorectal cancer (CRC). However, whether miR-133b is associated with CRC stemness and chemoresistance is not clear. In this study, we report that miR-133b is downregulated in colorectal spheroids, which are enriched with CSCs and display stem cell-like characteristics, including upreulation of CSCs surface markers and elevated chemoresistance. Additionally, miR-133b overexpression reduces CRC stemness and overrides chemoresistance to 5-Fluorouracil (5-FU) and oxaliplatin (OXP), indicating a negative role of miR-133b in regulating CRC stemness and chemoresistance. Moreover, miR-133b directly targets and suppresses the expression of disruptor of telomeric silencing 1-like (DOT1L), an exclusive H3K79 methyltransferase. Furthermore, miR-133b overexpression suppresses DOT1L-mediated H3K79me2 modification of stem cell genes, which is consistent with their downregulated transcription. More importantly, DOT1L restoration abrogates the suppressive effects of miR-133b on CRC stemness and chemoresistance, hence demonstrating that miR-133b regulates CRC stemness and chemoresistance through targeting DOT1L. Overall, these results imply that miR-133b might represent a novel therapeutic target in interfering CRC stemness and chemoresistance.

Loss of TRIM29 suppresses cancer stem cell-like characteristics of PDACs via accelerating ISG15 degradation.

TRIM family proteins are defined as E3 ubiquitin ligases because of their RING-finger domains. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, that is implicated in the posttranslational modification of diverse proteins. Both TRIM29 and ISG15 play both pro-tumoral and anti-tumoral functions in cancer cells derived from different histology. In the current study, we demonstrated that correlation expression of TRIM29 and ISG15 in pancreatic ductal adenocarcinomas (PDACs). The current study demonstrated that TRIM29 knockdown destabilized ISG15 protein via promoting its processing by calpain 3 (CAPN3). Importantly, the current study found that TRIM29 knockdown suppressed cancer stem cell-like features of PDACs, which can be rescued by ISG15 independent of its conjugation function. In addition, the current study demonstrated that extracellular free ISG15 played an important role in maintenance of cancer stem cell-like features of PDACs. Thereby, the current study displayed a novel mechanism by which TRIM29 modulates ISG15 stability via CAPN3-mediated processing, and subsequently extracellular ISG15 maintains the cancer stem cell-like features of PDAC via autocrine mode of action.

PLOD2 contributes to drug resistance in laryngeal cancer by promoting cancer stem cell-like characteristics

BackgroundAdvanced stage laryngeal squamous cell carcinoma (LSCC) presents a poor prognosis; thus, there is a great need to identify novel prognostic molecular markers. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) is thought to be a novel prognostic factor in several cancers, but its role in LSCC remains unknown. Cancer stem cells (CSCs) are responsible for most instances of tumor recurrence and the development of drug resistance and have been proven to be present in head and neck cancers. Our preliminary study indicated that PLOD2 was elevated in LSCC tissues; therefore, we hypothesized that PLOD2 is related to the prognosis of LSCC patients and aimed to explore the role and underlying mechanism of PLOD2 in LSCC.MethodsWe validated the prognostic role of PLOD2 in 114 LSCC patients by immunohistochemistry. Stable PLOD2-overexpressing Hep-2 and FaDu cells were established and assessed by molecular biology and biochemistry methods both in vitro and in vivo.ResultsWe confirmed that PLOD2 overexpression was correlated with poor prognosis in LSCC patients. PLOD2 overexpression strengthened the CSC-like properties of Hep-2 and FaDu cells, activated the Wnt signaling pathway and conferred drug resistance in LSCC in vitro and in vivo.ConclusionsWe found that PLOD2 could serve as a prognostic marker in patients with LSCC and confer drug resistance in LSCC by increasing CSC-like traits; in addition, a Wnt-responsive CSC pathway was identified.

Bone marrow microenvironment: The guardian of leukemia stem cells.

Bone marrow microenvironment (BMM) is the main sanctuary of leukemic stem cells (LSCs) and protects these cells against conventional therapies. However, it may open up an opportunity to target LSCs by breaking the close connection between LSCs and the BMM. The elimination of LSCs is of high importance, since they follow cancer stem cell theory as a part of this population. Based on cancer stem cell theory, a cell with stem cell-like features stands at the apex of the hierarchy and produces a heterogeneous population and governs the disease. Secretion of cytokines, chemokines, and extracellular vesicles, whether through autocrine or paracrine mechanisms by activation of downstream signaling pathways in LSCs, favors their persistence and makes the BMM less hospitable for normal stem cells. While all details about the interactions of the BMM and LSCs remain to be elucidated, some clinical trials have been designed to limit these reciprocal interactions to cure leukemia more effectively. In this review, we focus on chronic myeloid leukemia and acute myeloid leukemia LSCs and their milieu in the bone marrow, how to segregate them from the normal compartment, and finally the possible ways to eliminate these cells.

Transition from mesenchymal to bleb-based motility is predominantly exhibited by CD133-positive subpopulation of fibrosarcoma cells.

Metastatic disease is caused by the ability of cancer cells to reach distant organs and form secondary lesions at new locations. Dissemination of cancer cells depends on their migration plasticity - an ability to switch between motility modes driven by distinct molecular machineries. One of such switches is mesenchymal-to-amoeboid transition. Although mesenchymal migration of individual cells requires Arp2/3-dependent actin polymerisation, amoeboid migration is characterised by a high level of actomyosin contractility and often involves the formation of membrane blebs. The acquisition of amoeboid motility by mesenchymal cells is often associated with enhanced metastasis. We studied the ability of mesenchymal HT1080 fibrosarcoma cells to switch to amoeboid motility. We induced the transition from lamellipodium-rich to blebbing phenotype either by down-regulating the Arp2/3 complex, pharmacologically or by RNAi, or by decreasing substrate adhesiveness. Each of these treatments induced blebbing in a subset of fibrosarcoma cells, but not in normal subcutaneous fibroblasts. A significant fraction of HT1080 cells that switched to blebbing behaviour exhibited stem cell-like features, such as expression of the stem cell marker CD133, an increased efflux of Hoechst-33342 and positive staining for Oct4, Sox2 and Nanog. Furthermore, the isolated CD133+ cells demonstrated an increased ability to switch to bleb-rich amoeboid phenotype both under inhibitor's treatment and in 3D collagen gels. Together, our data show a significant correlation between the increased ability of cells to switch between migration modes and their stem-like features, suggesting that migration plasticity is an additional property of stem-like population of fibrosarcoma cells. This combination of features could facilitate both dissemination of these cells to distant locations, and their establishment self-renewal in a new microenvironment, as required for metastasis formation. These data suggest that migration plasticity is a new feature of cancer stem-like cells that can significantly facilitate their dissemination to a secondary location by allowing them to adapt quickly to challenging microenvironments. Moreover, it complements their resistance to apoptosis and self-renewal potential, thus enabling them not only to disseminate efficiently, but also to survive and colonise new niches.

Prooxidative Activity of Celastrol Induces Apoptosis, DNA Damage, and Cell Cycle Arrest in Drug-Resistant Human Colon Cancer Cells.

Cancer resistance to chemotherapy is closely related to tumor heterogeneity, i.e., the existence of distinct subpopulations of cancer cells in a tumor mass. An important role is assigned to cancer stem cells (CSCs), a small subset of cancer cells with high tumorigenic potential and capacity of self-renewal and differentiation. These properties of CSCs are sustained by the ability of those cells to maintain a low intracellular reactive oxygen species (ROS) levels, via upregulation of ROS scavenging systems. However, the accumulation of ROS over a critical threshold disturbs CSCs—redox homeostasis causing severe cytotoxic consequences. In the present study, we investigated the capacity of celastrol, a natural pentacyclic triterpenoid, to induce the formation of ROS and, consequently, cell death of the colon cancer cells with acquired resistant to cytotoxic drugs (LOVO/DX cell line). LOVO/DX cells express several important stem-like cell features, including a higher frequency of side population (SP) cells, higher expression of multidrug resistant proteins, overexpression of CSC-specific cell surface marker (CD44), increased expression of DNA repair gene (PARP1), and low intracellular ROS level. We found that celastrol, at higher concentrations (above 1 μM), significantly increased ROS amount in LOVO/DX cells at both cytoplasmic and mitochondrial levels. This prooxidant activity was associated with the induction of DNA double-strand breaks (DSBs) and apoptotic/necrotic cell death, as well as with inhibition of cell proliferation by S phase cell cycle arrest. Coincubation with NAC, a ROS scavenger, completely reversed the above effects. In summary, our results provide evidence that celastrol exhibits effective cytotoxic effects via ROS-dependent mechanisms on drug-resistant colon cancer cells. These findings strongly suggest the potential of celastrol to effectively kill cancer stem-like cells, and thus, it is a promising agent to treat severe, resistant to conventional therapy, colon cancers.