Induction Of Cancer Stem Cells Research Articles

Retinoids Induced Cancer Stem Cell Differentiation and Apoptosis for Cancer Therapies

Retinoids show great potential in various kinds of cancer chemotherapy due to its ability to induce signals for cell differentiation or death, as well as inhibit cancer stem cell proliferation. This paper summarized the recent progress of retinoids induced cancer stem cell differentiation and apoptosis in cancer therapy field, with the highlighted novel retinoid named WYC-209 in our lab, which could inhibit the tumor stem cell and malignant melanoma tumors with high efficacy and little toxicity.

HER2 decreases drug sensitivity of ovarian cancer cells via inducing stem cell-like property in an NFκB-dependent way.

Increasing evidence shows that cancer stem cells are responsible for drug resistance and relapse of tumors. In breast cancer, human epidermal growth factor receptor 2 (HER2) induces Herceptin resistance by inducing cancer stem cells. In the present study, we explored the effect of HER2 on cancer stem cells induction and drug sensitivity of ovarian cancer cell lines. First, we found that HER2 overexpression (HER2 OE) induced, while HER2 knockdown (HER2 KD) decreased CD44+/CD24− population. Consistently, HER2 expression was closely correlated with the sphere formation efficiency (SFE) of ovarian cancer cells. Second, we found that NFκB inhibition by specific inhibitor JSH23 or siRNA targetting subunit p65 dramatically impaired the induction of ovarian cancer stem cells by HER2, indicating that NFκB mediated HER2-induced ovarian cancer stem cells. Third, we found that HER2 KD significantly attenuated the tumorigenicity of ovarian cancer cells. Further, we found that HER2 inhibition increased drastically the sensitivity of ovarian cancer cells to doxorubicin (DOX) or paclitaxel (PTX). Finally, we examined the correlation between HER2 status and stem cell-related genes expression in human ovarian tumor tissues, and found that expressions of OCT4, COX2, and Nanog were higher in HER2 positive tumors than in HER2 negative tumors. Consistently, the 5-year tumor-free survival rate of HER2 positive patients was dramatically lower than HER2 negative patients. Taken together, our data indicate that HER2 decreases drug sensitivity of ovarian cancer cells via inducing stem cell-like property.

GSK3\u03b2 regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer

BackgroundTriple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC.MethodsA library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3β inhibitors were identified as EMT inhibitors. The effects of GSK3β inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3β correlates with the overall survival of breast cancer patients.ResultsWe identified a GSK3β inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3β inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3β reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3β inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24− in cells after exposure to GSK3β inhibitors. We found that GSK3β inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3β inhibitors and found that GSK3β inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3β correlated with poorer overall patient survival.ConclusionsTaken together, our data demonstrate that GSK3β is a potential target for TNBCs and suggest that GSK3β inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3β inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.

Loss of MYBBP1A Induces Cancer Stem Cell Activity in Renal Cancer.

Tumors are cellular ecosystems where different populations and subpopulations of cells coexist. Among these cells, cancer stem cells (CSCs) are considered to be the origin of the tumor mass, being involved in metastasis and in the resistance to conventional therapies. Furthermore, tumor cells have an enormous plasticity and a phenomenon of de-differentiation of mature tumor cells to CSCs may occur. Therefore, it is essential to identify genetic alterations that cause the de-differentiation of mature tumor cells to CSCs for the future design of therapeutic strategies. In this study, we characterized the role of MYBBP1A by experiments in cell lines, xenografts and human tumor samples. We have found that MYBBP1A downregulation increases c-MYB (Avian myeloblastosis viral oncogene homolog) activity, leading to a rise in the stem-like cell population. We identified that the downregulation of MYBBP1A increases tumorigenic properties, in vitro and in vivo, in renal carcinoma cell lines that express high levels of c-MYB exclusively. Moreover, in a cohort of renal tumors, MYBBP1A is downregulated or lost in a significant percentage of tumors correlating with poor patient prognosis and a metastatic tendency. Our data support the role of MYBBP1A as a tumor suppressor by repressing c-MYB, acting as an important regulator of the plasticity of tumor cells.

Sulforaphane Inhibits the Acquisition of Tobacco Smoke-Induced Lung Cancer Stem Cell-Like Properties via the IL-6/ΔNp63α/Notch Axis.

Background: Tobacco smoke (TS) critically contributes to the development of lung cancer; however, the underlying molecular mechanisms remain unclear. The induction of cancer stem cells (CSCs) by TS represents an early event in tumor initiation. The lung cancer-related gene ΔNp63α is highly expressed in epithelial tissues and drives tumor formation and cancer stem cell properties. This study investigated the role of ΔNp63α in the long-term acquisition of TS-induced lung CSC-like properties.Methods: The expression levels of ΔNp63α, lung CSC markers, and interleukin (IL)-6 in lung carcinoma specimens were determined by western blotting and enzyme linked immunosorbent assays. Human bronchial epithelial (HBE) cells were chronically exposed to 2 % cigarette smoke extract for 55 passages, following which colony formation capacity, expression of proteins associated with malignant transformation, lung CSC markers, and tumor incidence were investigated. The effects of ΔNp63α on long-term TS exposure-induced lung CSC-like properties and Notch activation were analyzed using tumorsphere formation ability, immunofluorescence assays, luciferase reporter assays, and western blotting. The roles of IL-6 on chronic TS exposure-induced lung CSC-like properties and ΔNp63α expression were also examined. Moreover, the effects of sulforaphane (SFN) on TS-transformed lung CSC-like properties, IL-6 and ΔNp63α expression, and Notch signaling were investigated in vitro and in vivo.Results: Higher levels of ΔNp63α were observed in the lung cancer tissues of smokers than in those of non-smokers, whereas ΔNp63α was positively correlated with CD133 and Oct4 expression in lung cancer tissues. Data from the in vivo and in vitro experiments demonstrated that long-term TS exposure-transformed HBE (THBE) cells acquired lung CSC-like properties. Furthermore, ΔNp63α transcriptionally activated the Notch signaling pathway to promote the acquisition of CSC-like properties by the THBE cells. TS upregulated IL-6, which increased ΔNp63α expression in THBE sphere-forming cells. Finally, SFN inhibited the TS-induced CSC-like properties of THBE cells via the IL-6/ΔNp63α/Notch axis.Conclusion: Our data suggest that the IL-6/ΔNp63α/Notch axis plays an important role in the long-term TS exposure-induced acquisition of lung CSC-like properties and SFN intervention.

The Chemokine Receptor CXCR3 Isoform B Drives Breast Cancer Stem Cells.

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24− phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target.

Mcl-1/CDK9 Targeting By AZD5991/AZD4573 Overcomes Intrinsic and Acquired Venetoclax Resistance in Vitro and In Vivo in PDX Model of AML through Modulation of Cell Death and Metabolic Functions

Mcl-1 and Bcl-2 are two major anti-apoptotic Bcl-2 proteins frequently overexpressed in malignant cells. They cooperatively support cell survival and are associated with therapy resistance. ABT-199 (venetoclax), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML as a single agent. Mcl-1 is induced by and a major resistance factor to ABT-199. Mcl-1 was recently found to also positively regulate mitochondrial oxidative phosphorylation that induces cancer stem cells and promotes chemoresistance. Mcl-1 is essential for the development of AML and for the survival of AML cells and stem cells. Increased mitochondrial oxidative phosphorylation has been demonstrated in these cells.First we found that Mcl-1 overexpressing (OE)/knockdown (KD) AML cells were markedly more resistant/sensitive to ABT-199 than were corresponding control cells, supporting the notion of Mcl-1 as a resistance factor to ABT-199. Inhibition of Mcl-1 by the selective Mcl-1 inhibitor AZD5991 or the CDK9 inhibitor AZD4573, which down-regulates short-lived proteins such as Mcl-1, induced apoptosis and showed strong synergy when combined with ABT-199 in AML cell lines, primary AML blasts, and stem/progenitor cells from patients. Importantly, combinations of AZD5991 or AZD4573 with ABT-199 synergistically induced apoptosis in OCI-AML3 and Mcl-1 OE cells intrinsically resistant to ABT-199 and in AML cell lines and primary patient cells with acquired resistance to ABT-199.Although OE/KD Mcl-1 in AML cells did not show obvious alterations in baseline cell viability, NSGS mice harboring Mcl-1 OE/KD OCI-AML3 cells survived significantly shorter/longer than those transplanted with control cells, supporting additional, non-apoptogenic functions of Mcl-1 in AML. We observed that genetic modulation of Mcl-1 alters cellular mitochondrial respiration and ROS levels. AML cells with Mcl-1 OE/KD increased/decreased O2 consumption and mitochondrial ATP and ROS generation. Consistent with this finding, inhibition of Mcl-1 by AZD5991 or AZD4573 decreased O2 consumption and ATP generation in AML cells and also in MV4-11 cells with acquired ABT-199 resistance. Mass spectrometry-based stable isotope tracing experiments using 1,2-13C-glucose showed that both genetic and pharmacological inhibition of Mcl-1 decreased flux of glucose carbon through glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting a role for Mcl-1 in cellular respiration and redox metabolism.To further assess the efficacy of combined Mcl-1 and Bcl-2 inhibition in primary AML cells resistant to ABT-199, we developed a PDX model using cells from an AML patient who initially responded to ABT-199/demethylating agent and then relapsed. NSGS mice engrafted with these PDX cells were treated with ABT-199 (50 mg/kg, oval gavage qd), AZD5991 (60 mg/kg, i.v. weekly), AZD4573 (15 mg/kg, i.p. bid with 2 h interval for two consequent days/week), ABT-199+AZD5991, or ABT-199+AZD4573 for 6 wks. Flow cytometric analysis of circulating human CD45+ cells on day 18 of therapy showed that each agent significantly decreased leukemia burden and that the combinations were significantly more effective (P<0.01) than each single agent. CyTOF analysis of BM cells (day 25) showed that both combinations markedly reduced (P<0.001) human CD45+ cells and, more importantly, human CD34+CD38+/CD38- and CD34+CD38+/CD38-CD123+ cells. Those combination treatments also decreased Mcl-1, Bcl-2, b-catenin, c-Myc, and FAK protein expression in CD34+CD38-CD123+ cells. Interestingly, AZD5991, AZD4573, or their combinations with ABT-199 greatly decreased CXCR4 in all cell populations. Ultimately, each single agent only marginally prolonged survival, whereas ABT-199+AZD4573 and even more so ABT-199+AZD5991 markedly improved survival in this highly ABT-199 resistant PDX model (Fig).Conclusion: we demonstrate that Mcl-1 has metabolic functions in AML and that inhibition of Mcl-1 enhances ABT-199 apoptogenic activity and overcomes intrinsic and acquired ABT-199 resistance in vitro and in vivo in a PDX murine model of AML, suggesting that inhibition of Mcl-1 improves the efficacy of ABT-199, and overcomes established resistance to Bcl-2 inhibition. Suppressing metabolic activity and CXCR4 inhibition may also contribute to the efficacy of this combination against AML stem cells in the BM microenvironment. DisclosuresCarter:AstraZeneca: Research Funding; novartis: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties. Cidado:AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment. Andreeff:AstraZeneca: Research Funding.

Prostaglandin E2 produced by myeloid-derived suppressive cells induces cancer stem cells in uterine cervical cancer.

Myeloid-derived suppressor cells (MDSCs) enhance tumor progression by suppressing tumor-specific T cell responses, stimulating tumor angiogenesis, or promoting tumor cell metastasis. However, the biology of MDSCs have not been fully investigated. In the current study, we investigated the role of MDSCs in inducing cancer stem-like cells and explored a clinically feasible approach for targeting MDSCs-mediated cancer stem-like cells induction. In vitro and in vivo experiments revealed that MDSCs induced by tumor-derived G-CSF enhanced the stemness of cervical cancer cells by producing Prostaglandin E2 (PGE2). We also demonstrated that anti-Gr-1 neutralizing antibody or celecoxib inhibited the induction of cancer stem-like cells and enhanced the efficacy of cisplatin in cervical cancer. In clinical samples, MDSCs, PGE2, and CSCs had positive correlations. In conclusion, G-CSF-induced MDSCs enhance the stemness of uterine cervical cancer cells by producing PGE2. Targeting MDSCs or PGE2 might be a reasonable strategy for enhancing the efficacies of treatments.

The inflammatory cytokine TNF contributes with RAC3-induced malignant transformation.

RAC3 is a coactivator of steroid receptors and NF-κB. It is usually overexpressed in several tumors, contributes to maintain cancer stem cells and also to induce them when is overexpressed in non-tumoral cells. In this work, we investigated whether the inflammatory cytokine TNF may contribute to the transforming effects of RAC3 overexpression in the non-tumoral HEK293 cell line. The study model included the HEK293 tumoral transformed cell line constitutively overexpressing RAC3 by stable transfection and control non-tumoral cells transfected with an empty vector. The HeLa and T47D tumoral cells that naturally overexpress RAC3 were used as positive control.We found that TNF potentiated RAC3-induced mesenchymal transition, involving an increased E-Cadherin downregulation, Vimentin and SNAIL upregulation and enhanced migratory behavior. Moreover, concerning the molecular mechanisms by which TNF potentiates the RAC3 transforming action, they involve the IKK activation, which in addition induced the β-Catenin transactivation. Our results demonstrate that although RAC3 overexpression could be a signal strong enough to induce cancer stem cells, the inflammatory microenvironment may be playing a key role contributing to the migratory and invasive phenotype required for metastasis and cancer persistence.

Cyclooxygenase-2 in cancer: A review.

Cyclooxygenase-2 (COX-2) is frequently expressed in many types of cancers exerting a pleiotropic and multifaceted role in genesis or promotion of carcinogenesis and cancer cell resistance to chemo- and radiotherapy. COX-2 is released by cancer-associated fibroblasts (CAFs), macrophage type 2 (M2) cells, and cancer cells to the tumor microenvironment (TME). COX-2 induces cancer stem cell (CSC)-like activity, and promotes apoptotic resistance, proliferation, angiogenesis, inflammation, invasion, and metastasis of cancer cells. COX-2 mediated hypoxia within the TME along with its positive interactions with YAP1 and antiapoptotic mediators are all in favor of cancer cell resistance to chemotherapeutic drugs. COX-2 exerts most of the functions through its metabolite prostaglandin E2. In some and limited situations, COX-2 may act as an antitumor enzyme. Multiple signals are contributed to the functions of COX-2 on cancer cells or its regulation. Members of mitogen-activated protein kinase (MAPK) family, epidermal growth factor receptor (EGFR), and nuclear factor-κβ are main upstream modulators for COX-2 in cancer cells. COX-2 also has interactions with a number of hormones within the body. Inhibition of COX-2 provides a high possibility to exert therapeutic outcomes in cancer. Administration of COX-2 inhibitors in a preoperative setting could reduce the risk of metastasis in cancer patients. COX-2 inhibition also sensitizes cancer cells to treatments like radio- and chemotherapy. Chemotherapeutic agents adversely induce COX-2 activity. Therefore, choosing an appropriate chemotherapy drugs along with adjustment of the type and does for COX-2 inhibitors based on the type of cancer would be an effective adjuvant strategy for targeting cancer.

Therapeutic potency of oncolytic virotherapy-induced cancer stem cells targeting in brain tumors, current status, and perspectives.

Brain tumors are the most common form of solid tumors in children and is presently a serious therapeutic challenge worldwide. Traditional treatment with chemotherapy and radiotherapy was shown to be unsuccessful in targeting brain tumor cancer stem cells (CSCs), leading to recurrent, treatment-resistant secondary malignancies. Oncolytic virotherapy (OV) is an effective antitumor therapeutic strategy which offers a novel, targeted approach for eradicating pediatric brain tumor CSCs by utilizing mechanisms of cell killing that differ from conventional therapies. A number of studies and some clinical trials have therefore investigated the effects of combined therapy of radiations or chemotherapies with oncolytic viruses which provide new insights regarding the effectiveness and improvement of treatment responses for brain cancer patients. This review summarizes the current knowledge of the therapeutic potency of OVs-induced CSCs targeting in the treatment of brain tumors for a better understanding and hence a better management of this disease.

Abstract A080: Caveolin-1 detected in tumor-derived exosomes induces cancer stem cell phenotypes in castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is by far the most common male malignancy and the third leading cause of cancer-related death in the US. Regardless of a great response of hormonal therapy for metastatic PCa patients, eventually PCa relapses to an end-stage castration-resistant prostate cancer (CRPC) leading to mortality. Although introducing newer androgen receptor (AR) pathway inhibitors is encouraging, treatment resistance in CRPC continues to remain a clinical problem. Clinical evidence of treatment resistant CRPC indicates carcinoma cell undergoing transdifferentiation. However, the mechanism as well as cell lineage are not fully characterized, which highlights an unmet clinical need to uncover potential therapeutic targets for targeted therapy to improve the overall survival of CRPC patients. Exosome is characterized as nano-grade small extracellular vesicle derived from the endosomal system from many kinds of cells. Therefore, exosome can carry many kinds of message molecules as a delivery vehicle in a paracrine or endocrine manner. Overall, tumor-derived exosomes (TDE) has been shown to play a critical role in promoting cancer progression or drug resistance. Our recent data have shown that TDE from PCa cells is able to induce neuroendocrine differentiation of CRPC, which leads us to further elucidate the role of PCa-derived exosome. Previously by using xenograft model, we have reported that elevated caveolin-1 (Cav-1) in LNCaP cell can accelerate its tumor growth but also increase the tumor incidence of low tumorigenic cells in a paracrine manner, implying that Cav-1 can be secreted extracellualrly. However, the functional role of secreted Cav-1 in PCa is still being debated. The expression of Cav-1 was considered to function as a suppressor of cell transformation and as preventing the development and metastases of mammary tumor; on the other hand, Cav-1 served as an oncogene that correlates with PCa progression. Furthermore, recent studies have shown that Cav-1 expression is associated with chemoresistance in multiple cancers as well as cancer stem cell (CSC) property in lung cancer. CSC theory has raised significant attention in recent years because of its self-renewal and quiescent nature, which underlies its role as a cancer-initiating cell population responsible for recurrent tumor with treatment resistance. In this study, we purified TDE from various PCa cell lines including either Cav-1 positive (Cav-1High) or negative (Cav-1low) cells using membrane-based affinity chromatography, and further characterized its size and number using Zetaview (Particle Matrix, Mebane, NC) and Exocet quantitation kit (SBI, Palo Alto, CA) separately. We found that TDE derived from metastatic PCa cells without AR expression is capable of inducing CSC phenotypes in AR-expressing recipient cells. Further analyses demonstrated that the high accumulation of Cav-1 in TDE appears to be a potential candidate. Indeed, Cav-1High cells or exogenous Cav-1-treated PCa cell lines can increase CSC phenotypes in vitro and in vivo. Mechanistically, the presence of Cav-1 is able to increase stem cell transcription Yamanaka factors, including KLF4, SOX2, OCT4, and Nanog. Furthermore, from microarray screening, we found RPC32 as another potential Cav-1 downstream effector, which is characterized as a subunit of RNA polymerase III (Pol III). RPC32 has two different isoforms (α and β), and only RPC32α is known to be highly regulated and expressed in stem cell and transformed cells. Therefore, we hypothesize that RPC32α is responsible for the elevated Yamanaka factors protein expression leading to CSC development. Nevertheless, we conclude that the presence of Cav-1 in PCa TDE plays an inductive role in the appearance of treatment-resistant CRPC in a paracrine manner, which provides a potential prognostic marker and therapeutic target. Citation Format: Chun-Jung Lin, Andrew Dang, Elizabeth Hernandez, Jer-Tsong Hsieh. Caveolin-1 detected in tumor-derived exosomes induces cancer stem cell phenotypes in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A080.

Abstract A73: Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7

Abstract Purpose: We aimed to understand how stemness is regulated by the Snail/let-7 axis in ovarian cancer cells, toward the goal of developing novel treatments that target stem-like cells to sensitize chemoresistant tumors. Background: Metastatic progression and disease recurrence in high-grade serous ovarian carcinoma (HGSOC) are in part driven by epithelial-mesenchymal transition (EMT) and cell reversion to cancer stem cell (CSC) phenotype. A contributor to the CSC phenotype is loss of miRNA let-7, due to increased expression of its targets, including oncogenes and pluripotency factors. Mechanisms leading to loss of let-7 are incompletely understood. Methods: Snail was overexpressed by estrogen receptor fusion, or EMT was induced with epidermal growth factor (EGF). Snail knockdown (KD) was by lentiviral small hairpin (sh)RNA expression and puromycin selection. Cell surface expression of CSC markers was analyzed by flow cytometry. Expression at the protein level was detected by immunofluorescence microscopy and Western blot. Gene expression of mesenchymal, epithelial, and pluripotency factors and miRNAs was detected by q-RT-PCR. Migration was determined by scratch assay and live-cell imaging. Data were analyzed by linear regression. Chromatin immunoprecipitation of Snail was followed by qPCR. Let-7i promoter luciferase with and without Snail were co-transfected into 293T cell lines and followed by luciferase assays for detection of bioluminescence. Let-7 overexpression was by transfection of mimics. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six-week-old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized HGSOC cells (OVCAR8 vs PDX) with control vs. Snail KD (250-500K cells). Bioluminescence was quantified by IVIS Lumina III over 21-50 days and analyzed by Living Image software. Results: We characterized several cell lines that accurately model HGSOC for their epithelial (E) vs. mesenchymal (M) and stem cell properties. OVSAHO, COV318, and OVCAR8 were selected for further study in increasing order of mesenchymal characteristics. Increasing let-7 levels or decreasing Snail levels disrupted the stem cell phenotype, reduced cells’ migratory ability, and sensitized cells to cisplatin. Snail bound promotors of let-7 in cell lines tested. Luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic tumor burden was significantly reduced in orthotopic xenografts using OVCAR8 cells expressing Snail shRNA. Similarly, in PDX derived from ascites or ovarian tumors, there was reduced tumor burden in Snail knockdown animals. Conclusions: We present preliminary data showing that the EMT factor Snail represses let-7, placing Snail at the nexus of dedifferentiation via loss of let-7, and invasiveness via EMT. PDX reproducibly phenocopy HGSOC. Cell line and patient-derived data support the relationship between Snail expression, let-7 downregulation, and the induction of CSC. We hypothesize that Snail KD disrupts the cancer stem cell state and disrupts disease progression. We propose that Snail is a potential pharmaceutical target for recurrent, metastatic EOC. Citation Format: Alyse Huisken, Nozomi Hojo, Hanmin Wang, Evgeny Chirshev, Carlotta Glackin, Yevgeniya Ioffe, Julia J. Unternaehrer-Hamm. Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A73.

Inhibition of GSK3 and MEK induced cancer stem cell generation via the Wnt and MEK signaling pathways.

Cancer stem cells (CSCs) are considered to be tumor-initiating cells, responsible for tumor invasive growth and dissemination to distant organ sites. Typically, radiation treatment and chemotherapy should target CSCs. However, current research investigating CSCs is impeded by the difficulty of isolating pure CSCs and maintaining them in vitro. In the present study, the synergistic inhibition of glycogen synthase kinase 3 and mitogen-activated protein kinase kinase using small molecules, CHIR99021 and PD184352, efficiently generated CSCs from immortalized human mammary epithelial cells (HMLEs) and resulted in the acquisition of mesenchymal traits and the expression of epithelial-mesenchymal transition markers. The cell proliferation, invasion and migration of HMLE cells were significantly promoted by CHIR99021 and PD184352 (P<0.05). Furthermore, the cell cycle was shifted from the G0/G1 phase to the G2/M phase, and the apoptotic rate was suppressed in HMLE cells following treatment with CHIR99021 and PD184352. Compared with control group, the stimulated cells exhibited an increased ability to form mammospheres and regenerate a tumor. In addition to these properties, the induced cells also exhibited notable chemotherapy resistance. In vivo, the treatment of cells with CHIR99021 and PD184352 promoted the growth of HMLE-engrafted tumor types. These results provide a practical strategy for the generation of CSCs using small molecules in vitro, which provides a cell resource that may be used for drug screening. Additionally, the present results additionally highlighted the synergistic functions of Wnt and mitogen-activated protein kinase kinase signaling pathways in tumorigenesis.

PO-300 Unveiling and exploiting cancer stem cell editing and immunogenicity for precision medicine

IntroductionImmunogenic chemotherapy (IC) induces immunogenic cell death (ICD), which, similar to viral infection, leads to a cancer-cell autonomous Type-I-Interferon (IFN-I) signalling. This immunological signature is crucial for effective antitumor responses but may paradoxically promote the emergence of a rare population of cancer stem cells (CSCs) acting as a chemoresistant niche within the tumour and roots for metastasis and relapse. In this study, we have investigated the role of IFN-I during IC in inducing a cancer editing program resulting in the appearance of poor immunogenic CSCs.Material and methodsHuman and murine tumour cell lines were treated in vitro with ICD-inducers or IFN-I as control and the induction of CSC were analysed by cytofluorometry, quantitative real time (qRT)-PCR, 3D culture and functional assays. Free and vesicle-mediated nucleic acid transfer during ICD has been characterised by co-culture experiments. IC-induced CSC immunogenicity has been studied through cytofluorometry, microfluidic devices and in vivo experiments. All experiments have been done in triplicate and statistical significance evaluated by two-tailed Student’s t test and two-way ANOVA.Results and discussionsThe transient/acute induction of IFN-I during ICD is followed by the appearance of a rare population of CSCs. Both free nucleic acids and extracellular vesicles are released during tumour ICD constituting the upstream inducers of IFN-I-mediated reprogramming of neighbouring cells. IC-induced CSCs display epithelial-to-mesenchymal transition traits, multidrug resistance and regenerative properties, and a significant tumorigenic potential when inoculated in immunodeficient and immunocompetent mice. As expected, tumour growth and size are reduced in the presence of an intact immune system. Experiments on microfluidic devices reveal a poor immunogenic potential of CSCs, further confirmed by the expression of immune checkpoint blockers.ConclusionOur results pinpoint a surprising link between ICD, IFN-I and CSCs. Elucidating the mechanisms of CSC editing together with a deep characterisation of CSC (immune) properties could be crucial to prevent tumour relapse. This could undoubtedly have dramatic implications for the clinical management of cancer in an era of terrific development of precision combined chemo-immune therapy.

Abstract 1989: Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7

Abstract Purpose:We aimed to understand how stemness is regulated by the Snail/let-7 axis in ovarian cancer cells, toward the goal of developing novel treatments that target stem-like cells to prevent recurrence. Experimental Procedures:We determined the correlation between mesenchymal and stem cell phenotype in a panel of high grade serous ovarian cancer (HGSOC) cells, including patient-derived cells. Cell surface expression of cancer stem cell (CSC) markers was analyzed by flow cytometry. Expression at the protein level was detected by Western blot, at RNA level by q-RT-PCR. Epithelial-mesenchymal transition (EMT) was induced with epidermal growth factor (EGF). Snail knockdown (KD) was achieved by lentiviral small hairpin (sh)RNA expression and puromycin selection. Migration was determined by wound healing assay, chemoresistance by MTT assay. Chromatin immunoprecipitation of Snail was followed by qPCR. Let-7i promoter luciferase with and without Snail were co-transfected into 293T cell lines and followed by luciferase assays for detection of bioluminescence. Let-7 overexpression was by transfection of miRNA mimics. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six week old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized HGSOC cells (OVCAR8 vs PDX) with control vs. Snail KD. Bioluminescence was quantified by IVIS Lumina III. Results:We characterized several cell lines that accurately model HGSOC for their epithelial (E) vs. mesenchymal (M) and stem cell properties using a novel index. OVSAHO, Kuramochi, COV318, and OVCAR8 were selected for further study in increasing order of M characteristics. M cell status correlated with morphology, stemness, and invasiveness. Increasing the tumor suppressor miRNA let-7 levels or decreasing Snail levels disrupted CSC phenotype, reduced cells' migratory ability, and sensitized cells to cisplatin. Snail bound promotors of let-7 , and luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic tumor burden was significantly reduced in orthotopic xenografts using OVCAR8 cells expressing Snail shRNA. Similarly, in PDX derived from ascites or ovarian tumors, there was reduced tumor burden in Snail KD animals. Conclusions:The EMT factor Snail represses let-7, placing Snail at the nexus of dedifferentiation via loss of let-7, and invasiveness via EMT. Orthotopic PDX demonstrate that Snail targeting reduces tumor burden. Cell line and patient-derived data supports the relationship between Snail expression, let-7 downregulation, and the induction of CSC. We hypothesize that Snail expression destabilizes differentiation and introduces the CSC state in part via let-7 repression. We propose that Snail is a potential target for recurrent, metastatic EOC. Citation Format: Nozomi Hojo, Alyse Huisken, Hanmin Wang, Evgeny Chirshev, Sang Nguyen, Carlotta Glackin, Yevgeniya Ioffe, Juli Unternaehrer. Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7 [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 1989.

Abstract 4494: HMGA1 drives tumor progression and recruits cancer-associated fibroblasts in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors for which there are no effective therapies. Emerging evidence suggests that the tumor stroma interacts with the cancer cells to induce cancer stem cell properties and drive tumor progression. In PDAC, the fibroblast stroma also provides a dense barrier preventing cytotoxic therapy from reaching PDAC cells. We previously discovered that high levels of High Mobility Group A1 (HMGA1) protein predict decreased survival in primary PDAC. Here, we report a novel role for HMGA1-FGF19 in mediating tumor-stromal interactions and tumor progression. Silencing HMGA1 in PDAC cell lines or low-passge, patient-derived cells abruptly halts proliferation. Spindle-shaped, mesenchymal cells became reprogrammed into cuboidal, more epithelial-like cells. Sensitivity to gemcitabine was enhanced and colony formation, migration, invasion, and three-dimensional (3D) sphere formation were all disrupted in cells with HMGA1 knock-down. Silencing HMGA1 also disrupted xenograft tumorigenesis and depleted cancer stem cells/tumor-initiator cells in limiting dilution tumorigenicity assays. To elucidate underlying molecular mechanisms mediating these striking phenotypes, we performed RNA-seq after silencing HMGA1 in invasive, highly metastatic, low-passage patient-derived PDAC cells (10.7). Among the genes regulated by HMGA1 were those encoding proteins involved in tumor-stromal signaling, including the fibroblast growth factor 19 (FGF19). The FGF19 gene is highly expressed in GI tumors (liver, colon, PDAC) and transgenic mice overexpressing Fgr15 (the murine homolog) in hepatocytes develop hepatocellular carcinoma (HCC). FGF19 also correlates with poor outcomes in human HCC and colon cancer, although it's role in PDAC was unknown. Here, we found that FGF19 expression is dependent upon HMGA1 in 3 different PDAC cell lines; silencing HMGA1 represses FGF19 in these cells. HMGA1 also binds directly to the FGF19 promoter at 2 predicted DNA binding sites as assessed by chromatin immunoprecipiation. To determine whether FGF19 plays a functional role in HMGA1-mediated tumor progression and cancer stem cell properties, we silenced FGF19 in PDAC cells. Similar to our results with HMGA1, silencing FGF19 impaired PDAC growth and 3D sphere formation in vitro. Because fibroblast growth factors interact with fibroblasts, we determined whether the HMGA1-FGF19 pathway was involved in tumor cell – stromal crosstalk. PDAC 10.7 cells recruit cancer-associated fibroblasts (CAFs) in a co-culture system, although this recruitment was abrogated when HMGA1 was silenced. CAF migration was also disrupted by anti-human FGF19 neutralizing antibodies. Together, these findings indicate that HMGA1 drive tumor progression and cancer stem cell properties through FGF19 and suggest that targeting the HMGA1-FGF19 pathway maybe efficaceous in PDAC. Citation Format: Shuai Shuai, Lingling Xian, Tait Huso, Karen Reddy, Linda Resar. HMGA1 drives tumor progression and recruits cancer-associated fibroblasts in pancreatic ductal adenocarcinoma [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 4494.

Abstract 769: Zeb-1 contributes to the development of chemotherapeutic resistance in mantle cell lymphoma

Abstract Despite progress overall in improving cancer treatments, the complete response of mantle cell lymphoma (MCL) is still limited due to the inevitable development of drug resistance. Understanding how MCL cells acquire resistance at the molecular level may be a key innovation to targeted therapy. We established a series of bortezomib (BTZ)-resistant MCL cells and performed gene/protein profiling along with metabolic fingerprinting using synchrotron-based Fourier-transform infrared spectroscopy. Zeb-1, an epithelial-mesenchymal transition-inducing transcription factor better known for promoting metastasis in solid carcinomas, was found to be aberrantly expressed in the resistant MCL cells in accordance with an excessive production of lipids. Ectopic expression of Zeb-1 in parental MCL cells rendered the cells to acquire apoptosis resistance to BTZ. Here we demonstrated, for the first time, two possible encompassing mechanisms for Zeb-1-mediated BTZ resistance: (i) Zeb-1 induces cancer stem cells, as evaluated by tumor spheres and aldehyde dehydrogenase activity; and (ii) Zeb-1 induces lipid reprograming that may interfere with cell growth and drug response. Together, our results unveil a novel role of Zeb-1 in MCL aggressiveness. These findings have a clinical significance insight to the development of chemotherapeutic resistance of MCL. Citation Format: Sudjit Luanpitpong, Jirarat Poohadsuan, Montira Janan, Kanjana Thumanu, Yon Rojanasakul, Surapol Issaragrisil. Zeb-1 contributes to the development of chemotherapeutic resistance in mantle cell lymphoma [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 769.

Chronic ethanol exposure of human pancreatic normal ductal epithelial cells induces cancer stem cell phenotype through SATB2.

The incidence of pancreatic cancer is on the rise. Risk factors for pancreatic cancer include alcohol toxicity and metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance and type 2 diabetes. However, the molecular mechanism by which chronic alcohol consumption contributes to pancreatic cancer is not well understood. The purpose of the study was to demonstrate the effects of long‐term chronic ethanol exposure on the transformation of human pancreatic normal ductal epithelial (HPNE) cells. Our data showed that ethanol‐transformed HPNE cells were more progressively transformed exhibiting spheroids and colonies, and anchorage‐independent growth. These transformed cells contained high levels of reactive oxygen species and induced SATB2 expression. Furthermore, during ethanol‐induced cellular transformation, cells gained the phenotypes of cancer stem cells (CSCs) by expressing pluripotency maintaining factors (Oct4, Sox2, cMyc and KLF4) and stem cell markers (CD24, CD44 and CD133). Ethanol‐induced SATB2 can bind to the promoters of KLF4, Oct4, cMyc, Sox2, Bcl‐2 and XIAP genes. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibited cell proliferation, colony formation and markers of CSCs and pluripotency. These data suggest that chronic alcohol consumption may contribute toward the development of pancreatic cancer by converting HPNE cells to cancer stem‐like cells.

CXCL12γ Promotes Metastatic Castration-Resistant Prostate Cancer by Inducing Cancer Stem Cell and Neuroendocrine Phenotypes.

There is evidence that cancer stem-like cells (CSC) and neuroendocrine behavior play critical roles in the pathogenesis and clinical course of metastatic castration-resistant prostate cancer (m-CRPC). However, there is limited mechanistic understanding of how CSC and neuroendocrine phenotypes impact the development of m-CRPC. In this study, we explored the role of the intracellular chemokine CXCL12γ in CSC induction and neuroendocrine differentiation and its impact on m-CRPC. CXCL12γ expression was detected in small-cell carcinoma of metastatic tissues and circulating tumor cells from m-CRPC patients and in prostate cancer cells displaying an neuroendocrine phenotype. Mechanistic investigations demonstrated that overexpression of CXCL12γ induced CSC and neuroendocrine phenotypes in prostate cancer cells through CXCR4-mediated PKCα/NFκB signaling, which promoted prostate tumor outgrowth, metastasis, and chemoresistance in vivo Together, our results establish a significant function for CXCL12γ in m-CRPC development and suggest it as a candidate therapeutic target to control aggressive disease.Significance: Expression of CXCL12γ induces the expression of a cancer stem cell and neuroendocrine phenotypes, resulting in the development of aggressive m-CRPC. Cancer Res; 78(8); 2026-39. ©2018 AACR.