Fraction Of Cancer Stem Cells Research Articles

Cancer Cell CD44 Mediates Macrophage/Monocyte-Driven Regulation of Head and Neck Cancer Stem Cells.

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.

The Role of Breast Cancer Stem Cell-Related Biomarkers as Prognostic Factors.

Breast cancer is one of the leading causes of cancer-related deaths in women worldwide, and its incidence is on the rise. A small fraction of cancer stem cells was identified within the tumour bulk, which are regarded as cancer-initiating cells, possess self-renewal and propagation potential, and a key driver for tumour heterogeneity and disease progression. Cancer heterogeneity reduces the overall efficacy of chemotherapy and contributes to treatment failure and relapse. The cell-surface and subcellular biomarkers related to breast cancer stem cell (BCSC) phenotypes are increasingly being recognised. These biomarkers are useful for the isolation of BCSCs and can serve as potential therapeutic targets and prognostic tools to monitor treatment responses. Recently, the role of noncoding microRNAs (miRNAs) has extensively been explored as novel biomarker molecules for breast cancer diagnosis and prognosis with high specificity and sensitivity. An in-depth understanding of the biological roles of miRNA in breast carcinogenesis provides insights into the pathways of cancer development and its utility for disease prognostication. This review gives an overview of stem cells, highlights the biomarkers expressed in BCSCs and describes their potential role as prognostic indicators.

C-KIT regulates stability of cancer stemness in CD44-positive colorectal cancer cells

Cancer stem cells (CSCs) are subpopulations of cancer cells with high self-renewal potential that are involved in tumor progression and recurrence. It has been postulated that CSCs and non-stem cancer cells are inter-convertible. However, precise mechanisms for the plasticity and stability of cancer stemness remain elusive. Here, we demonstrate that CD44-positive colorectal CSC fractions contain two types of cancer cells: “CD44-stable” cells, in which CD44 expression is stably sustained, and “CD44-trasnsient” cells, which are rapidly converted to CD44-negative cells. CD44-stable cells expressed higher levels of c-KIT tyrosine kinase than CD44-transient cells. c-KIT knockdown by siRNAs converted the CD44-positive cells to CD44-negative cells, which expressed lower levels of stem cell markers such as ASCL2 and EPCAM. In the CD44-positive cells, c-KIT phosphorylation level was very low whereas stem cell factor, a c-KIT ligand, elevated c-KIT phosphorylation without affecting stem cell marker expression. CRISPR-Cas9-mediated knockout of the c-KIT gene in CD44 stable cells attenuated the CSC properties including expression of CD44 and other stem cell markers, clonogenicity and in vivo tumorigenic potential in a mouse xenograft model. These observations suggest that the colorectal CSC fractions contain cancer cells with differential plasticity, which is determined by c-KIT.

In Silico Models Accurately Predict In Vivo Response for IL6 Blockade in Head and Neck Cancer.

Malignant features of head and neck squamous cell carcinoma (HNSCC) may be derived from the presence of stem-like cells that are characterized by uniquely high tumorigenic potential. These cancer stem cells (CSC) function as putative drivers of tumor initiation, therapeutic evasion, metastasis, and recurrence. Although they are an appealing conceptual target, CSC-directed cancer therapies remain scarce. One promising CSC target is the IL6 pathway, which is strongly correlated with poor patient survival. In this study we created and validated a multiscale mathematical model to investigate the impact of cross-talk between tumor cell- and endothelial cell (EC)-secreted IL6 on HNSCC growth and the CSC fraction. We then predicted and analyzed the responses of HNSCC to tocilizumab (TCZ) and cisplatin combination therapy. The model was validated with in vivo experiments involving human ECs coimplanted with HNSCC cell line xenografts. Without artificial tuning to the laboratory data, the model showed excellent predictive agreement with the decrease in tumor volumes observed in TCZ-treated mice, as well as a decrease in the CSC fraction. This computational platform provides a framework for preclinical cisplatin and TCZ dose and frequency evaluation to be tested in future clinical studies. SIGNIFICANCE: A mathematical model is used to rapidly evaluate dosing strategies for IL6 pathway modulation. These results may lead to nonintuitive dosing or timing treatment schedules to optimize synergism between drugs.

Cross-Talk Between circRNAs and mRNAs Modulates MiRNA-mediated Circuits and Affects Melanoma Plasticity.

CircularRNAs (circRNAs) are non-coding RNAs which compete for microRNA (miRNA) binding, influencing the abundance and stability of other RNA species. Herein we have investigated the effect of circRNAs on the mir200-ZEB1 feedback loop in relationship with the aggressiveness of human melanoma cells. We first compared the level of expression of key factors in the mir200-ZEB1 feedback loop in primary human melanoma cells compared with their matching metastatic one and found a correlation between the aggressiveness of the cells and the level of expression of ZEB1 and SNAI1. We also analyzed factors in the mir200-ZEB1 feedback loop, including circZEB1, during the phenotypic switching of human melanoma cells. Our results showed a correlation between the level of ZEB1 and SNAI1 and the fraction of cancer stem cells in the population. The level of circZEB1 was, however, consistently high during the entire phenotypic transformation. To understand this result we propose a mathematical model of the regulatory circuit. According to the model, the experimental observations can be explained by the presence of a back-splicing factor limiting circRNA production.

Dichloroacetate (DCA) and Cancer: An Overview towards Clinical Applications.

An extensive body of literature describes anticancer property of dichloroacetate (DCA), but its effective clinical administration in cancer therapy is still limited to clinical trials. The occurrence of side effects such as neurotoxicity as well as the suspicion of DCA carcinogenicity still restricts the clinical use of DCA. However, in the last years, the number of reports supporting DCA employment against cancer increased also because of the great interest in targeting metabolism of tumour cells. Dissecting DCA mechanism of action helped to understand the bases of its selective efficacy against cancer cells. A successful coadministration of DCA with conventional chemotherapy, radiotherapy, other drugs, or natural compounds has been tested in several cancer models. New drug delivery systems and multiaction compounds containing DCA and other drugs seem to ameliorate bioavailability and appear more efficient thanks to a synergistic action of multiple agents. The spread of reports supporting the efficiency of DCA in cancer therapy has prompted additional studies that let to find other potential molecular targets of DCA. Interestingly, DCA could significantly affect cancer stem cell fraction and contribute to cancer eradication. Collectively, these findings provide a strong rationale towards novel clinical translational studies of DCA in cancer therapy.

Hypoxia-induced secretion stimulates breast cancer stem cell regulatory signalling pathways.

It is well known that tumour cells are dependent on communication with the tumour microenvironment. Previously, it has been shown that hypoxia (HX) induces pronounced, diverse and direct effects on cancer stem cell (CSC) qualities in different breast cancer subtypes. Here, we describe the mechanism by which HX‐induced secretion influences the spreading of CSCs. Conditioned media (CM) from estrogen receptor (ER)‐α‐positive hypoxic breast cancer cell cultures increased the fraction of CSCs compared to normal growth conditions, as determined using sets of CSC assays and model systems. In contrast, media from ERα‐negative hypoxic cell cultures instead decreased this key subpopulation of cancer cells. Further, there was a striking overrepresentation of JAK‐STAT‐associated cytokines in both the ERα‐positive and ERα‐negative linked hypoxic responses as determined by a protein screen of the CM. JAK‐STAT inhibitors and knockdown experiments further supported the hypothesis that this pathway is critical for the CSC‐activating and CSC‐inactivating effects induced by hypoxic secretion. We also observed that the interleukin‐6‐JAK2‐STAT3 axis was specifically central for the ERα‐negative hypoxic behaviour. Our results underline the importance of considering breast cancer subtypes in treatments targeting JAK‐STAT or HX‐associated processes and indicate that HX is not only a confined tumour biological event, but also influences key tumour properties in widespread normoxic microenvironments.

Central role of IP3R2-mediated Ca2+ oscillation in self-renewal of liver cancer stem cells elucidated by high-signal ER sensor

Ca2+ oscillation is a system-level property of the cellular Ca2+-handling machinery and encodes diverse physiological and pathological signals. The present study tests the hypothesis that Ca2+ oscillations play a vital role in maintaining the stemness of liver cancer stem cells (CSCs), which are postulated to be responsible for cancer initiation and progression. We found that niche factor-stimulated Ca2+ oscillation is a signature feature of CSC-enriched Hep-12 cells and purified α2δ1+ CSC fractions from hepatocellular carcinoma cell lines. In Hep-12 cells, the Ca2+ oscillation frequency positively correlated with the self-renewal potential. Using a newly developed high signal, endoplasmic reticulum (ER) localized Ca2+ sensor GCaMP-ER2, we demonstrated CSC-distinctive oscillatory ER Ca2+ release controlled by the type 2 inositol 1,4,5-trisphosphate receptor (IP3R2). Knockdown of IP3R2 severely suppressed the self-renewal capacity of liver CSCs. We propose that targeting the IP3R2-mediated Ca2+ oscillation in CSCs might afford a novel, physiologically inspired anti-tumor strategy for liver cancer.

Ablation of Cancer Stem Cells by Therapeutic Inhibition of the MDM2-p53 Interaction in Mucoepidermoid Carcinoma.

Unique cells characterized by multipotency, self-renewal, and high tumorigenic potential have been recently discovered in mucoepidermoid carcinomas. These cells are defined by high aldehyde dehydrogenase activity and high CD44 expression (ALDHhighCD44high) and function as cancer stem cells (CSC). It has been recently shown that p53 regulates cell differentiation, suggesting that induction of p53 by therapeutic blockade of the MDM2-p53 interaction may constitute a novel strategy to ablate CSCs. Here, we evaluated the effect of a small-molecule inhibitor of MDM2-p53 interaction (MI-773) on the fraction of CSCs in mucoepidermoid carcinoma. Human mucoepidermoid carcinoma cells (UM-HMC-1,-3A,-3B) were used to assess the effect of MI-773 on cell survival, cell cycle, fraction of CSCs, and expression of p53, p21, MDM2, and Bmi-1 (key regulator of self-renewal). Mice bearing xenograft tumors generated with these mucoepidermoid carcinoma cells were treated with MI-773 to determine the effect of MDM2-p53 inhibition on CSCs in vivo. MDM2 is highly expressed in human mucoepidermoid carcinoma tissues. MI-773 induced expression of p53 and its downstream targets p21 and MDM2, caused G1 cell-cycle arrest, and induced mucoepidermoid carcinoma tumor cell apoptosis in vitro. Importantly, a marked decrease in expression of Bmi-1 and in the fraction of ALDHhighCD44high (CSCs) was caused by MI-773 in vitro and in mice harboring mucoepidermoid carcinoma xenografts. Collectively, these data demonstrate that MI-773 reduces the fraction of CSCs, suggesting that patients with mucoepidermoid carcinoma might benefit from therapeutic inhibition of the MDM2-p53 interaction.

PARP Inhibition Induces Enrichment of DNA Repair-Proficient CD133 and CD117 Positive Ovarian Cancer Stem Cells.

PARP inhibitors (PARPi) are FDA-approved monotherapy agents for the treatment of recurrent ovarian cancer in patients with and without a BRCA mutation. Despite promising response rates, not all patients derive benefit, and the majority develop resistance. PARPi treatment in vitro and in vivo induced an enrichment of CD133+ and CD117+ ovarian cancer stem cells (CSC). This effect was not affected by BRCA mutation status. In the CSC fractions, PARPi induced cell-cycle arrest in G2-M with a consequent accumulation of γH2AX, RAD51, and uniquely DMC1 foci. DNA damage and repair monitoring assays demonstrated that CSCs display more efficient DNA repair due, in part, to activation of embryonic repair mechanisms which involved the RAD51 homologue, DMC1 recombinase. Preserved and induced homologous repair (HR) could be a mechanism of an inherent resistance of CSCs to the synthetic lethality of PARPi that likely promotes disease recurrence. IMPLICATIONS: Treatment with PARPi fails to significantly affect ovarian cancer CSC populations, likely contributing to recurrent disease. Ovarian cancer CSCs stabilize genomic integrity after PARPi treatment, due to a more efficient inherent DNA repair capacity. PARPi-induced DMC1 recombinase and HR proficiency provide CSCs the opportunity to repair DNA damage more efficiently.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/2/431/F1.large.jpg.

Toward understanding cancer stem cell heterogeneity in the tumor microenvironment

The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) formation are two paramount processes driving tumor progression, therapy resistance, and cancer metastasis. Recent experiments show that cells with varying EMT and CSC phenotypes are spatially segregated in the primary tumor. The underlying mechanisms generating such spatiotemporal dynamics in the tumor microenvironment, however, remain largely unexplored. Here, we show through a mechanism-based dynamical model that the diffusion of EMT-inducing signals such as TGF-β, together with noncell autonomous control of EMT and CSC decision making via the Notch signaling pathway, can explain experimentally observed disparate localization of subsets of CSCs with varying EMT phenotypes in the tumor. Our simulations show that the more mesenchymal CSCs lie at the invasive edge, while the hybrid epithelial/mesenchymal (E/M) CSCs reside in the tumor interior. Further, motivated by the role of Notch-Jagged signaling in mediating EMT and stemness, we investigated the microenvironmental factors that promote Notch-Jagged signaling. We show that many inflammatory cytokines such as IL-6 that can promote Notch-Jagged signaling can (i) stabilize a hybrid E/M phenotype, (ii) increase the likelihood of spatial proximity of hybrid E/M cells, and (iii) expand the fraction of CSCs. To validate the predicted connection between Notch-Jagged signaling and stemness, we knocked down JAG1 in hybrid E/M SUM149 human breast cancer cells in vitro. JAG1 knockdown significantly restricted tumor organoid formation, confirming the key role that Notch-Jagged signaling can play in tumor progression. Together, our integrated computational-experimental framework reveals the underlying principles of spatiotemporal dynamics of EMT and CSCs.

Deep Tumor-Penetrated Nanocages Improve Accessibility to Cancer Stem Cells for Photothermal-Chemotherapy of Breast Cancer Metastasis.

Cancer stem cells (CSCs) are proposed to account for the initiation of cancer metastasis, but their accessibility remains a great challenge. This study reports deep tumor‐penetrated biomimetic nanocages to augment the accessibility to CSCs fractions in tumor for anti‐metastasis therapy. The nanocages can load photothermal agent of 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindotricarbocyanine iodide (DBN) and chemotherapeutic epirubicin (EBN) to eradicate CSCs for photothermal‐chemotherapy of breast cancer metastasis. In metastatic 4T1‐indcued tumor model, both DBN and EBN can efficiently accumulate in tumor sites and feasibly permeate throughout the tumor mass. These biomimetic nanosystems can be preferentially internalized by cancer cells and effectively accessed to CSCs fractions in tumor. The DBN+laser/EBN treatment produces considerable depression of primary tumor growth, drastically eradicates around 80% of CSCs fractions in primary tumor, and results in 95.2% inhibition of lung metastasis. Thus, the biomimetic nanocages can be a promising delivery nanovehicle with preferential CSCs‐accessibility for effective anti‐metastasis therapy.

Abstract 925: In silico models accurately predict in vivo response for IL-6 blockade in head and neck cancer

Abstract The purpose of this study is to create and validate a mathematical model describing the effect of interleukin-6 (IL-6) on head and neck cancer growth and cancer stem cell fraction. Head and Neck Squamous Cell Carcinoma (HNSCC) is the seventh most-common solid tumor with about 55,000 new cases diagnosed every year. Malignant features of HNSCC cells are derived from a shift towards more stem-like features, as postulated by the cancer stem cell (CSC) hypothesis. CSCs encompass a unique cellular subpopulation characterized by multipotency, uniquely high tumorigenic potential, and self-renewal. These cells function as putative drivers of tumor initiation, therapeutic evasion, metastasis, and recurrence. Though they are an appealing conceptual target, CSC directed cancer therapies remain scarce. High levels of both serum IL-6 and tumor IL-6 receptor (IL-6R) expression are strongly correlated with poor patient survival. Endothelial cell-secreted IL-6 enhances the tumorigenic potential and self-renewal of head and neck CSCs. Furthermore, interruption of the IL-6 pathway results in a decreased fraction of HNSCC CSCs. Efficacy and optimal administration of the FDA approved rheumatoid arthritis IL-6R antibody Tocilizumab in HNSCC is not known. Here we used a multi-scale mathematical model that operates at the intracellular, molecular, and tissue level to investigate the impacts of endothelial cell-secreted IL-6 signaling on the crosstalk between tumor cells and ECs during tumor growth. This endothelial cell - tumor cell (EC-TC) model was used to study the effect of tocilizumab treatment on HNSCC, and particularly the CSC fraction. Our ordinary differential equation model is the first of its kind to include full occupancy dynamics between endothelial-cell produced IL-6, IL-6R, and the competitive IL-6R inhibitor Tocilizumab. In order to biologically validate the utility of our mathematical model, we performed a series of in-vivo experiments using HNSCC cell lines and human endothelial cells co-embedded in a porous scaffold and implanted in SCID mice flank. Tumor volumes were measured serially. CSC fraction was determined in HNSCC cell lines using flow cytometry for aldehyde dehydrogenase (ALDH) high/CD44 high proportion. Without artificial tuning to the laboratory data, our model provided superb predictive agreement to the decrease in tumor volumes observed in TCZ treated mice (r = 0.947, p < 0.0001), as well as a decrease in CSC fraction. This predictive in-silico framework can serve to rapidly evaluate dosing strategies for IL-6 pathway modulation, as well as providing the basis for proposing combination treatments with IL-6 blockade and cytotoxic or other targeted therapies. Citation Format: Fereshteh Nazari, Alexandra E. Oklejas, Jacques E. Nör, Alexander T. Pearson, Trachette L. Jackson. In silico models accurately predict in vivo response for IL-6 blockade in head and neck cancer [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 925.

Abstract 3148: p140Cap affects breast cancer features through the regulation of the tumor microenvironment composition

Abstract Increasing data indicate the key role of the tumor microenvironment in cancer progression, metastasis formation and treatment sensitivity, where the tumor stroma and the immune infiltrate strongly influence tumor features. Our recent studies indicate that the adaptor protein p140Cap is highly expressed in about 50% of ERBB2-amplified breast cancer patients. Its expression correlates with increased patient survival and decreased probability to develop metastasis. Causally, p140Cap negatively regulates breast tumor properties both in vitro and in the NeuT preclinical mouse model. In this study, we observed an indirect influence of p140Cap-expression on tumor immune infiltrate composition of a syngeneic ERBB2+ breast cancer model. p140Cap tumor cells and tumors show reduced level of G-CSF mRNA and protein. Accordingly, we observed a reduced mobilization of G-CSF-dependent myeloid-derived suppressor cells (MDSCs- CD11b+Ly6G+Ly6Clo) from bone marrow and a decreased level of MDSCs in tumor, blood and spleen of mice orthotopically injected with p140Cap tumor cells, compared to controls. This “less” immune suppressive microenvironment associated to p140Cap-expression, was also confirmed by an increase in CD8+, M1 macrophages and Natural Killer lymphocytes concomitant with a decrease in M2 macrophages and FoxP3 regulatory T cells. Interestingly, the expression of p140Cap in cells and tumors significantly reduce the fraction of cancer stem cells and their sphere and tumor initiation frequency, by modulating their proliferative potential. Since that the tumor initiating cell exhibit an enhanced G-CSF production, these data suggest that p140Cap, by regulating the cancer stem cell compartment, limits ERBB2 tumor aggressiveness by creating a tumor microenvironment less favorable to tumor progression. Citation Format: Vincenzo Salemme. p140Cap affects breast cancer features through the regulation of the tumor microenvironment composition [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 3148.

PO-238 Dichloroacetate (DCA) treatment affects mitochondrial activity and stemness in pancreatic cancer (PC) cell lines

IntroductionTargeting metabolism represents a new approach to treat cancer, expecially when conventional chemoterapy fails. In this study, we tested a metabolic approach to treat PC, investigating, in vitro and in vivo, its response to DCA treatment.Material and methodsTwo PC cell lines, BXPC3 and PANC1, were treated with DCA 4 and 10 mM for 24 hour. Cell viability and proliferation were assessed by MTS assay and xCELLigence, apoptosis and ROS by flow-cytometry; pPDHSer293/tot PDH, LC3B, DRP1, MFN1, MNF2, OPA1 and TOMM20 protein expression was evaluated by western blotting, lin28 gene expression by qPCR. The oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) were measured by Seahorse Technology. Ultra-low attachment plates were used to form spheroids. In vivo, DCA was administered to BXPC3-luc tumor-bearing nude mice. After measuring bioluminescence signalling, the tumour masses were harvested, photographed and weighed.Results and discussionsDCA treatment reduced cell proliferation, decreasing cell survival with an increase in ROS production and apoptosis in both cell lines. Despite PDH activation by dephosphorylation, DCA did not restore bioenergetic profile but decreased OCR, a measure of oxidative phosphorylation efficiency. ECAR was not affected, suggesting that the glicolytic capacity was not modified by DCA treatment. These observations led us to explore mitophagy, whose activation was confirmed by LC3B protein overexpression and TOMM20 downregulation, and mitochondrial dynamics also altered following DCA treatment as shown by the downregulation of MFN1, MFN2, OPA1, key proteins of mitochondrial fusion. Interestingly, DCA was able to negatively affect the cancer stem cell (CSCs) fraction in both cell lines, reducing the expression of stemness genes such as Lin28 and inhibiting spheroid formation. When added to 3D cultures already formed, it was able to downregulate stemness genes expression, leading to a significant size reduction and affecting spheroid viability. Finally, DCA efficacy was confirmed in a xenograft pancreatic cancer mouse model in which DCA treatment displayed a significant retarded progression of PC, reducing diameter of the tumour mass.ConclusionOur data suggest that DCA is able to strongly affect PC cells metabolism counteracting mitochondrial activity. This effect is not related to PDH activity stimulation. In addition, the ability of DCA to hit CSCs offers a further rationale to candidate this drug for PC treatment, trying to reach a complete tumour eradication.

Targeting of BMI-1 expression by the novel small molecule PTC596 in mantle cell lymphoma.

Despite the development of the novel Bruton tyrosine kinase inhibitor ibrutinib, mantle cell lymphoma (MCL) remains an incurable B-cell non-Hodgkin lymphoma. BMI-1 is required for the self-renewal and maintenance of MCL-initiating stem cells. Upregulation of BMI-1 has been reported in MCL patients, especially in those with refractory/relapsed disease. We studied the effects of a novel small-molecule selective inhibitor of BMI1 expression, PTC596, in MCL cells. Eight MCL cell lines and patient-derived samples were exposed to PTC596. PTC596 induced mitochondrial apoptosis, as evidenced by loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation, and phosphatidylserine externalization. There was a positive correlation between baseline BMI-1 protein levels and PTC596-induced apoptosis. p53 status did not affect sensitivity to PTC596. PTC596 effectively decreased BMI-1-expressing and tumor-initiating side population MCL cells (IC50: 138 nM) compared with ibrutinib, which modestly decreased side population cells. Interestingly, PTC596, reported to target cancer stem cells, decreased MCL-1 expression levels and antagonized ibrutinib-induced increase in MCL-1 expression, leading to synergistic apoptosis induction in MCL cells. There are currently no drugs that specifically target cancer stem cell fractions, and a reduction in BMI-1 protein by PTC596 may offer a novel therapeutic strategy for MCL.

TBX21 predicts prognosis of patients and drives cancer stem cell maintenance via the TBX21\u2013IL-4 pathway in lung adenocarcinoma

BackgroundThe Th1 cell-specific transcription factor TBX21 functions as a regulator of expression of a Th1 cytokine, interferon gamma (IFN-γ). However, the specific function of TBX21 correlated with cancer stemness remains unclear.MethodsUsing univariate and multivariate survival analysis, TBX21was identified as an independent predictive factor and was associated with poor prognosis in 1389 patients with lung adenocarcinoma (LUAD). Its mechanism in the prognosis was explored by functional enrichment analysis and validated in bioexperiments.ResultsIn the training and test sets, TBX21 could classify 1389 LUAD patients into high and low-risk groups with significantly different prognosis (P < 0.01). Its prognostic power was independent of other clinical factors including stage, age, gender and smoking status. Functional studies indicated that downregulating TBX21 in lung cancer cells decreased the fraction of cancer stem cells and their sphere and tumor initiation frequency. Furthermore, the study showed that TBX21 activation transduced a TBX21–IL-4 signaling cascade to promote tumor initiation, tumor growth and expression of stemness markers.ConclusionsThese data demonstrated a key role of TBX21 in the maintenance of cancer stemness and that the TBX21–IL-4 pathway is a crucial factor contributing to lung carcinogenesis.Graphical abstractTBX21 prognostic model correlated with cancer stemness via TBX21-IL-4 pathway in LUAD patients

Abstract B042: Anticancer effects of processed garlic constituent diallyl trisulfide on head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite significant advances in therapeutic approaches, survival rate is less than 50%. Early detection and treatment will help in prevention of spreading of invasive cancer and thereby improve the survival rates. Studies showed that dietary intake of Allium vegetables lowered the risk of various cancers. Studies reported DATS as a promising chemopreventive constituent of processed garlic that induced cell cycle arrest and apoptosis in prostate and breast cancer. However, it has not been investigated for its efficacy and molecular mechanisms of chemopreventive effects of DATS against HNSCC. Herein, we investigated the effect of DATS on HNSCC FaDu, D562, UMSCC-22A, UMSCC-22B, and Cal33 cells in culture. DATS (10-100 µM) treatment significantly reduced the growth, proliferation, and survival of HNSCC cells. We observed that DATS treatments significantly induced G2/M cell cycle arrest. DATS treatment resulted in the accumulation of cyclin B1 and induced the levels of cip1/p21. DATS-induced mitotic arrest was attenuated by NAC pretreatment, which suggested that DATS mediates growth-suppressive effects by induction of ROS. DATS induced cell death through apoptosis, which was associated with ROS production. Further, DATS increased the ratio of Bax/Bcl2 and cleavage of caspase-3 and PARP and decreased the levels of XIAP. Exposure of HNSCC cells to pharmacologic concentrations of DATS (1-10 μM) resulted in inhibition of cancer stem cells (CSC) fraction as evidenced by CD133high/CD44high fraction of cells, aldehyde dehydrogenase 1 (ALDH1) activity, and spheroid formation. DATS-treated tumor xenograft also revealed its in vivo inhibitory effect on CSCs. We observed for the first time that DATS treatment decreased tumor incidence and inhibited tumor promotion and progression in 4-Nitroquinoline 1-oxide (4-NQO)-induced mouse oral carcinogenesis model. Taken together, DATS strongly inhibited cell proliferation, induced cell cycle arrest, cell death via apoptosis, mitochondrial dysfunction and ROS generation and reduced CSC fraction, and decreased tumor incidence, promotion, and progression in 4-NQO model. Citation Format: Sivapar Mathan, Shivendra Singh, Rana P. Singh. Anticancer effects of processed garlic constituent diallyl trisulfide on head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B042.

Радиосенсибилизация опухолевых стволовых клеток аденокарциномы молочной железы человека линии MCF-7 с помощью никлозамида

Purpose: To investigate the effect of niclosamide on the radiosensitivity of MCF-7 cancer stem cells (CSC) and to explore the molecular mechanism of radiosensitization.&#x0D; Material and methods: Cancer stem cells were obtained from MCF-7 mammary adenocarcinoma monolayer culture (2D) and propagated as mammospheres (3D) in serum-free DMEM/F12 supplemented with EGF, bFGF, B 27, heparin, and insulin in ultra-low attachment plates. The single cell suspensions were irradiated with 1, 2, 4, 6, and 8 Gy using 60Co source at a dose rate of 0.54 Gy/min. Controls were sham irradiated. The size of the CD44+/CD24-/low CSC fraction was measured using flow cytometry at the 7th and 14th days after irradiation. The radiosensitizing effect of niclosamide on the 2D- and 3D-cell cultures was assessed by the number of cells at the 7th and 14th days after irradiation with doses of 2 or 4 Gy. Niclosamide was added to the cell cultures in a dose of 2 µM 1 h before irradiation. Media with niclosamide were changed to corresponding media without niclosamide 24 h post-irradiation. Cell survival was evaluated by hemocytometer cell counting using trypan blue. The size of the CSC fraction was measured at the 7th day after the irradiation with or without niclosamide. To investigate the effect of niclosamide on DNA damage the level of histone γH2AX was measured by flow cytometry at 60 min after irradiation.&#x0D; Results: Culturing adherent MCF-7 cells line under non-adherent mammosphere-forming conditions led to significantly increase in CSCs proportion. CD44+/CD24–/low cell fraction in 2D-culture was 0.2 ± 0.1 %, and it was 3.2 ± 0.6 % in 3D-culture (the third-generation spheres). Subpopulation of CSCs increased with the increase in irradiation dose. At day 14 after irradiation with the dose of 8 Gy, CD44+/CD24–/low cell fraction in 2D-culture was 2.2 ± 0.6 %, and it was 12.0 ± 0.9 % in 3D-culture. Cells grown in mammosphere-culture were found to have a higher survival rate after irradiation at doses of 2 and 4 Gy than cells grown in adherent conditions. At day 7 after irradiation with 2 and 4 Gy the 3D-culture cells counts decreased less than the 2D-culture cells counts (92 ± 13 % and 61 ± 10 % versus 58 ± 4 % and 37 ± 3 % compared to non-irradiated control). Moreover, cells grown in mammosphere-culture showed a higher renewal capacity. At day 14 after irradiation with 2 and 4 Gy the number of 3D-culture cells was 66 ± 11 % and 30 ± 7 % while the number of adherent cells was 43 ± 2 % and 7 ± 1 % compared to non-irradiated control. It was found that niclosomide has radiosensitizing effect on cells grown in mammospheres. Pretreatment with niclosamide 1 h prior to irradiation resulted in decrease in cell survival. At day 7 after irradiation at 2 and 4 Gy in the presence of niclosamide, the numbers of 3D-culture cells were reduced by 13 % and 7 % compared to their numbers after irradiation with appropriate doses in the absence of niclosamide. Furthermore, niclosamide was shown to reduce the percentage of CSCs. At day 7 after irradiation with 2 and 4 Gy, 3D-culture showed elevated percentages of CD44+/CD24−/low cells: CSC fraction in mammospheres were 4.6 ± 0.3 % and 6.1 ± 0.5 %, respectively, and after pretreatment by niclosamide and irradiation the sizes of the CSC fractions were 2.5 ± 0.1 % and 4.0 ± 0.3 %, respectively. Radiosensitizing effect of niclosamide lasted for 2 weeks. At day 14 after irradiation at 2 and 4 Gy in combination with niclosamide the numbers of 3D-culture cells were reduced by 13 % and 9 % compared to cell numbers after irradiation with appropriate doses without of niclosamide. Radiosensitization of cells grown in mammospheres might be related with increase in DNA double-strand breaks (DSB) prodused by niclosomide. At 1 h after 2 and 4 Gy irradiation cells grown in mammospheres shown 2.4-fold and 4.8-fold increase in DNA DSB, as indicated by γH2AX levels, compared to baseline. The combined treatment of niclosamide and irradiation with 2 and 4 Gy resulted in 3.4-fold and 5.5-fold increase in the levels of histone γ-H2AX, respectively.&#x0D; Conclusion: Niclosamide increased the radiosensetivity not only of the bulk cells of mammary adenocarcinoma line MCF-7 but also of CSCs. The radiosensitizing effect of niclosamide was associated with increase in the level of DNA damage.

Abstract A21: Inhibition of Sonic Hedgehog Pathway by small molecule inhibitors: Targeting colon cancer stem cells

Abstract The existence of Cancer Stem Cells (CSCs) has been proven extensively in recent years. CSCs have been positively identified in, and isolated from colon cancer. They have been found to be resilient to therapy and are highly metastatic and it has been suggested that this could be attributed to the presence of CSCs expressing cell surface CD133. An active Hedgehog-Gli pathway has been detected in human colon carcinoma cells as well as their stem cells. Sonic Hedgehog (Shh) inhibitors such as cyclopamine have been successful in the treatment of other cancer types. CSCs were isolated from HT29 and DLD1 cells using the CD133 surface protein and these cells were further characterized via their expression of key stem cell markers using fluorescence microscopy. These markers included c-Myc, KLF4 and alkaline phosphatase, all of which showed elevated expression levels in the CSC fraction. The growth pattern of CSCs in comparison to non-CSCs was analyzed using cell-impedance (xCELLigence) assays, with clear differences noted in their growth rates. Adhesion assays revealed that although CSCs have a lower proliferation rate, their adhesive potential proved to be greater, possibly due to them being highly metastatic. The effects of the Shh inhibitors cyclopamine and SANT-2 on cellular adhesion of colon CSCs were compared to the Shh pathway control molecule tomatidine, and a marked decrease in adhesion potential was noted. With regard to cell invasion, this was significantly decreased when CSCs were treated with the small molecule inhibitors. In cell migration/scratch assays a similar trend was seen, wherein cell migration was retarded by Hh pathway inhibition. Using confocal microscopy, the cell surface expression of Shh and CD133 was observed following treatment with cyclopamine and while found to significantly reduce Shh expression, CD133 expression remained unaffected. Considering these in vitro results small molecule inhibitors targeting the Shh pathway appear to be promising therapeutic tools for the treatment of metastatic colon CSCs. Citation Format: Aadilah Omar, Clement Penny, Paul Ruff. Inhibition of Sonic Hedgehog Pathway by small molecule inhibitors: Targeting colon cancer stem cells [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A21.