Human Cancer Stem Research Articles

Abstract 6067: Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines

Abstract Background: Our overarching goal is to therapeutically sensitize human colorectal cancer stem cells to differentiation-inducing effects of retinoid agents. Tumorigenesis in colorectal cancer (CRC) results from the acquisition of mutations in a colonic stem cell which transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC due to the resistance of CSCs to chemotherapy and/or radiation. All-trans retinoic acid (ATRA) is known to be a greatly effective treatment for acute promyelocytic leukemia (APL) which is caused by translocation in the retinoic acid receptor alpha (RARA) gene. We conjecture that the response of retinoic acid (RA) agents may have effects on solid tumors depending on their pathway genotype. Results from our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that the majority of RA pathway genes are overexpressed in CRC. Therefore, we hypothesize that the ability of retinoid and retinoid metabolism blocking agents to induce differentiating effects in CSCs depends on the RA pathway genotype expressed in CRC cells. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole and Talarozole) to inhibit proliferation and induce differentiation on CRC cell lines with different mutations in RA signaling were evaluated. Additionally, Nanostring Profiling was used to measure the effects of the agents on mRNA expression of CSC markers, indicators of cell differentiation, and RA receptors. Results: Our results show that the RA pathway genotype affects the response of RA agents in CRC cell lines. Both HCT116 and SW480 CRC cell lines, which are both mutant in RARA and RARA and RXRG respectively displayed resistance to ATRA. HT29 CRC cells express wild- type RA receptors and are sensitive to ATRA. All three cell lines showed similar dose responses to the CYP26A1 inhibitor Liarozole. This indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism increases intracellular RA levels in a similar manner on all 3 cell lines. Nanostring Profiling shows that ATRA treatment of HT29 cells induces: an increase in the expression of RARA, CYP26A1 (ATRA metabolizing enzyme) and KRT20 (differentiation marker); and a decrease in the expression of CSC markers LGR5 and ALDH1A1. This indicates an active RA signaling pathway, induced differentiation, and a decrease in stem cells. Discussion: Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Therefore, further investigation on how to therapeutically sensitize human colonic CSCs to the differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6067.

Abstract 3171: ARL2 is required for homologous recombination repair and colon cancer stem cell maintenance

Abstract ARL2 is a small GTP binding protein mediates many cytosolic functions including localization of Ras, dynamics of microtubule and fission of mitochondria but its nuclear function is not well elucidated yet. Here we present the novel role of ARL2 in human cancer nucleus and cancer stem cells (CSC). The cancer tissue gene expression data in The Human Protein Atlas and The Cancer Genome Atlas showed possible pro-cancer and nuclear role of ARL2 in colon cancers. The single cell RNA seq analysis suggests its function in colon stem cells. The activation mutant of K-Ras harboring colon cancer cells expressed less ARL2 than the wild type cells but increased ARL2 expression in CSC spheres. The deprivation of ARL2 in colon cancer cell lines using RNAi preferentially eliminated the CSC spheres while it resulted in minimal impact on bulk cultured cell (BCC) numbers. Suppression of ARL2 halted the BCC at M phase as expected but did not change cell cycle in CSC spheres. However, it led to accumulation of a DNA double strand break marker and apoptosis only in CSC spheres which suggest that ARL2’s requirement for double strand DNA break repair in CSC. Out of 6 somatic RAD51 family genes which all mediate the critical early events of homologous recombination repair (HRR), 5 showed significant and positive association with ARL2 in gene expression in human colon cancer tissue suggesting functional link between ARL2 and HRR. Indeed, ARL2 was required for HRR when double strand DNA breaks were induced. These data collectively showed that ARL2 may contribute to the HRR in the nucleus and the ARL2 mediated HRR is more critical for colon CSC survival. Citation Format: Seok Gyeong Choi, Hani Lee, Sojung Ha, Sukjoon Yoon, Woo-Young Kim. ARL2 is required for homologous recombination repair and colon cancer stem cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3171.

Lipophilic dye-compatible brain clearing technique allowing correlative magnetic resonance/high-resolution fluorescence imaging in rat models of glioblastoma

In this work we optimized a novel approach for combining in vivo MRI and ex vivo high-resolution fluorescence microscopy that involves: (i) a method for slicing rat brain tissue into sections with the same thickness and spatial orientation as in in vivo MRI, to better correlate in vivo MRI analyses with ex-vivo imaging via scanning confocal microscope and (ii) an improved clearing protocol compatible with lipophilic dyes that highlight the neurovascular network, to obtain high tissue transparency while preserving tissue staining and morphology with no significant tissue shrinkage or expansion. We applied this methodology in two rat models of glioblastoma (GBM; U87 human glioma cells and patient-derived human glioblastoma cancer stem cells) to demonstrate how vital the information retrieved from the correlation between MRI and confocal images is and to highlight how the increased invasiveness of xenografts derived from cancer stem cells may not be clearly detected by standard in vivo MRI approaches. The protocol studied in this work could be implemented in pre-clinical GBM research to further the development and validation of more predictive and translatable MR imaging protocols that can be used as critical diagnostic and prognostic tools. The development of this protocol is part of the quest for more efficacious treatment approaches for this devastating and still uncurable disease. In particular, this approach could be instrumental in validating novel MRI-based techniques to assess cellular infiltration beyond the macroscopic tumor margins and to quantify neo-angiogenesis.

Abstract 694: PAWI-2 overcomes tumor stemness and drug resistance in pancreatic cancer stem cells

Abstract Today, pancreatic cancer (PC) remains a major health problem in the US and soon will be the second most common cause of mortality due to cancer. However, most of the front-line PC chemotherapies cause serious side effects and a majority of patients with PC are often resistant to clinical therapies. The fact that cancer stem cells (CSCs) become enriched after anti-cancer therapy implicates CSCs as key contributors to tumor dormancy, metastasis, and relapse in PC. Given the important role of human pancreatic cancer stem cells (hPCSCs) in PC, development of a novel treatment strategy that targets hPCSCs or their extrinsic and intrinsic regulators could be of great clinical utility to treat PC. Herein, we used a highly validated hPCSCs model (FGβ3 cells) to test a novel compound (PAWI-2) to target CSCs. In this hPCSC model, integrin αvβ3 expression was shown to be a marker/functional contributor to CSC progression and drug resistance attributed to its ability to trigger anchorage-independent cell survival and tumor metastasis. Specifically, integrin αvβ3 recruits KRAS and RalB to tumor cell plasma membranes to activate NF-κB signaling that triggers dysregulated KRAS-RalB-NF-κB pathways. This was shown to be a pharmacological target to reverse CSC-like properties or re-sensitize drug resistance (e.g., EGFR inhibitors, erlotinib) of established FGβ3 tumors. Compared to parental FG cells, PAWI-2 showed greater potency to inhibit cell viability and self-renewal capacity of FGβ3 cells. PAWI-2 inhibited dysregulated integrin β3-KRAS-RalB-NF-κB signaling independent of KRAS and Ral. This is clinically relevant because >90% of KRAS is activated by mutations in PC and RAS or Ral inhibitors have not proven effective clinically. PAWI-2 targeted downstream of KRAS signaling and was negatively regulated by other feedback mechanisms. This was further shown by genetic knockdown of key regulator and co-treatment of specific inhibitors. PAWI-2 overcomes erlotinib resistance in FGβ3 cells with greater potency than bortezomib. In the proposed mechanism of PAWI-2 action, key regulators link the inhibition of KRAS signaling and cell cycle arrest (G2/M). The great utility and pharmacological potency of PAWI-2 in FGβ3 cells (that possess stem-like properties) compared to FG cells provide a basis for further development. PAWI-2 as an efficient therapeutic to reverse tumor stemness and resensitize CSC tumors to drug inhibition. This work was supported by Inception Award from California Institute for Regenerative Medicine (CIRM) (DISC1-10583; J.R. Cashman) and by funds from the Human BioMolecular Research Institute. Citation Format: Jiongjia Cheng, John Cashman, Jorge Gomez-Galeno. PAWI-2 overcomes tumor stemness and drug resistance in pancreatic cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 694.

Abstract B05: Basic research and translational medicine of bladder cancer in China

Abstract Over the past decade, the basic medical research has developed rapidly in China, and more attention is focused on the clinical application of scientific discoveries, resulting in increased contribution to human science and technology. Like other major diseases, the basic research and translational medicine of bladder cancer has been significantly improved, and the clinical translational research also showed a promising tendency. Zhiming Cai's team launched the “Genitourinary Tumor Genome Project” and established a biologic sample bank and biologic repositories for genitourinary tumors in 2010. The Digital Hospital for Bladder Cancer is also under construction. Based on the CRISPR-Cas9, our team synthesized the logical “AND” gate genetic circuits of bladder cancer cells. With discovered targets, the engineered cells were developed and applied to CAR-T and TCR-T clinical trials of bladder cancer. About 70 patients from the world were treated and satisfying clinical results were achieved. One patient with recurrent bladder cancer had stopped recurring for more than 3 years after TCR-T treatment. Song Wu's team has made great progress in bladder cancer stem cell research and the urine test for early detection of bladder cancer. Jian Huang's team launched the “Chinese Bladder Cancer Consortium” in 2013 and organized an annual national academic conference where hundreds of participants came together to expand the research of bladder cancer in China. Tianxin Lin's team identified a predictive marker for VEGF-C antibody therapy, providing a systematic solution for lymphatic metastasis of bladder cancer. Fangjian Zhou's team confirmed the high expression of exosomal circPRMT5 is a new biomarker for predicting lymphatic metastasis. Zusen Fan's team focused on the regulation of stem cell origin of bladder cancer, urine cell cytology diagnosis, and immunotherapy based on antibody BCMab1. Pingchou Han and Baiye Jin's team developed a novel microfluidic approach for detection of bladder cancer cells. Tingyi Wen and Chong Li's team identified a novel signal, the KMT1A-GATA3-STAT3 circuit, which promoted the self-renewal and tumorigenicity of human bladder cancer stem cells. Dingwei Ye and Jiejie Xu's team reveal the immunologic characteristics of tumors in the Chinese bladder cancer population. Also, Ye's team found a rare polymorphism in the UHRF1BP1 gene that was related to the bladder cancer incidence in Chinese. Jun Yan's team revealed overexpression of Wnt7a activated the Wnt/β-catenin signaling, which may induce the metastasis of bladder cancer. Zhengdong Zhang's team identified a new locus site of intron CWC27, which was associated with bladder cancer risk. Zhiping Wang's team found that the variants of SNPs in multiple DNA repair genes as well as some haplotypes were highly related with the bladder cancer, of which two locus sites of XRCC3 gene showed significant relation with the bladder cancer occurring in the Han population of Northwest China. Note: This abstract was not presented at the conference. Citation Format: Zhiming Cai. Basic research and translational medicine of bladder cancer in China [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B05.

Abstract LB-110: Proteasome addiction a new therapeutic opportunity to treat PTEN-deficient brain tumors

Abstract One of the most common genetic alterations in glioblastoma (30-40%) occurs in the tumor suppressor gene PTEN (phosphatase and tensin homolog), where loss of function has been mechanistically linked to increased tumor cell invasion, and to a lack of radio- and chemo-therapy response. To identify new drug compounds that target PTEN-deficient brain tumors we performed a high throughput drug screening using patient-derived GBM spheres and found that PTEN-deficient samples were highly sensitive to proteasome inhibitors. We confirmed PTEN dependency to proteasome inhibition by genetically overexpressing or deleting PTEN in GBM cells and established that the drug inhibition response relied on PTEN enzymatic activity. Additionally, proteasome inhibition specifically suppressed tumor growth and increased survival of mice orthotopically engrafted with human PTEN-null glioblastoma cancer stem cells. Mechanistically, we determined that PTEN-deficient cells were more sensitive to proteasome inhibition due to an increase in protein synthesis rate and loss of autophagy activity associated with activation of the PI3K/mTOR pathway. Finally, integrated in silico analysis of the Samsung Medical Center dataset corroborated that samples with PTEN/EGFR/PI3K alterations were significantly more sensitive to Carfilzomib, an FDA-approved proteasome inhibitor. This study reveals that PTEN-deficient cells are "proteasome addictive" and opens a new therapeutic opportunity to treat glioblastoma PTEN-deficient patients. Citation Format: Jorge A. Benitez, Darren Finlay, Alexandre Rosa-Campos, Jianhui Ma, Tomoyuki Koga, Kristiina Vuori, Frank Furnari. Proteasome addiction a new therapeutic opportunity to treat PTEN-deficient brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-110.

Sa1695 – Evaluating the Therapeutic Role of Metformin on Differentiation and Tumor Microenvironment of Human Colorectal Cancer Stem Cells In Vitro

Sa1695 – Evaluating the Therapeutic Role of Metformin on Differentiation and Tumor Microenvironment of Human Colorectal Cancer Stem Cells In Vitro

Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells

Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells. Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group, Nc-shRNA group and Notch1-shRNA group. The Nc-shRNA group was a negative control RNAi lentivirus group, and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group. The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1. The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay. Apoptosis was detected by Annexin V/7-AAD double staining. Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1. Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group, Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000±0.000, 0.937±0.025, 0.490±0.036 and 1.000±0.000, 1.077±0.070, 0.373±0.038, with statistically significant differences (F=359.707, P<0.001; F=210.455, P<0.001), further paired comparison, the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P<0.05). Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results. MTT assay showed that the 24 h A values of A549 cells in control group, Nc-shRNA group and Notch1-shRNA group were 0.209±0.005, 0.219±0.009, 0.159±0.006, 48 h A values were 0.293±0.004, 0.302±0.004, 0.205±0.005, 72 h A values were 0.450±0.003, 0.430±0.012, 0.348±0.017, with statistically significant differences (F=79.487, P<0.001; F=508.664, P<0.001; F=57.156, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24, 48, 72 h (all P<0.05). The 48 h A values of SPC-A-1 cells in control group, Nc-shRNA group and Notch1-shRNA group were 0.438±0.022, 0.412±0.015, 0.364±0.010, 72 h A values were 0.540±0.016, 0.519±0.009, 0.438±0.019, with statistically significant differences (F=15.667, P=0.004; F=37.299, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P<0.05). The sphere sizes of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667±6.506)μm, (136.667±7.095)μm, (86.676±7.638)μm, with statistically significant difference (F=65.940, P<0.001). The sphere sizes of the three groups in SPC-A-1 cells were (118.667±6.658)μm, (128.000±7.000)μm, (60.675±4.509)μm, with statistically significant difference (F=105.372, P<0.001). Further paired comparison, the sphere size of Notch1-shRNA group was significantly smaller than that of Nc-shRNA group in the two kinds of cells (all P<0.05). The apoptosis rates of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were (0.489±0.014)%, (0.633±0.021)%, (1.683±0.221)% and (1.323±0.194)%, (1.690±0.188)%, (3.017±0.356)%, with statistically significant differences (F=77.660, P<0.001; F=32.200, P=0.001), further paired comparison, the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P<0.05). Western blotting showed that the expressions of PCNA, Bcl-2 and Hes-1 in control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F=155.343, P<0.001; F=22.576, P=0.002; F=70.108, P<0.001), and the expressions of PCNA, Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F=49.419, P<0.001; F=28.090, P=0.001; F=12.040, P=0.007). Further paired comparison, the expressions of PCNA, Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells, and the differences were statistically significant (all P<0.05). Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis, which may be related to the down-regulation of its downstream gene Hes-1. Key words: Receptor, Notch1; Neoplastic stem cells; Carcinoma, non-small-cell lung; Cell proliferation; Apoptosis

MicroRNA-122 negatively associates with peroxiredoxin-II expression in human gefitinib-resistant lung cancer stem cells

Previously, we demonstrated that Prx II is important for survival of the gefitinib-resistant A549 (A549/GR) cell line, an NSCLC cell line derived by repeated exposure to gefitinib. Therefore, in this study, we used A549/GR cells to investigate the role of Prx II in GR NSCLC stemness. Initially, to explore the stemness characteristics and investigate the association of Prx II with those stemness characteristics, we successfully isolated a stem cell-like population from A549/GR cells. A549/GR CD133+ cells possessed important cancer stemness characteristics, including the abilities to undergo metastasis, angiogenesis, self-renewal, and to express stemness genes and epithelial–mesenchymal transition (EMT) markers. However, those characteristics were abolished by knocking down Prx II expression. MicroRNA 122 (miR-122) targets Prx II in A549/GR cancer stem cells (CSCs), thereby inhibiting the stemness characteristics in vitro and in vivo. Next, we investigate whether miR-122 overexpression was associated with Prx II expression and Prx-II-induced stemness characteristics, we transfected miR-122 into A549/GR CSCs. MiR-122 inhibited A549/GR stemness by downregulating the Hedgehog, Notch, and Wnt/β-catenin pathways. Taken together, our data suggest that Prx II promotes A549/GR stemness, and that targeting Prx II and miR-122 is a potentially viable strategy for anti-cancer-stem cell therapy in GR NSCLCs.

Single-cell evolution and molecular characterization of human colorectal cancer stem cells.

e15699Background: Tumor heterogeneity and internal clonal evolution of human colorectal cancer stem cells (CRCSCs) within cancerous tissues often represents an aggressive feature of cancer progress...

A gene expression signature to characterize human lung adenocarcinoma cancer stem cells.

e20547Background: Treatment resistance is linked to cancer stem cells (CSCs), a highly tumorigenic subpopulation of cells with the ability to grow as spheres in non-adherent conditions. The aim of ...

Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco’s Modified Eagle’s Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

Abstract 81: E2F4/p107 complex regulates chemotherapy resistance in human colorectal cancer stem cells

Abstract Most cancer patients experience disease progression despite one or multiple lines of treatment with cytotoxic chemotherapy. Evidence from human trials and in vivo experiments on patient-derived xenograft (PDX) tumor lines indicates that cancer stem cells (CSCs) can survive chemotherapy treatment. However, the mechanism of chemotherapy resistance in CSCs is not fully elucidated. To identify new mechanisms of the drug-resistance in colorectal cancer, we used gene-expression microarrays to examine the gene expression profile of CSCs and non-CSCs in four human PDX models of colorectal cancer, both before-and-after treatment with cytotoxic chemotherapy. The gene-expression data were analyzed using Independent Component Analysis (ICA) followed by Transcription Factor Target Genes (TFTG) enrichment analysis, which identified the transcription factor E2F4 as a candidate regulator of chemotherapy resistance in colorectal CSCs. E2F4 was predominantly expressed in the CSC-enriched cell population (CD44+/CD166+ human colorectal cancer cells). E2F4 knock-down sensitizes cancer cells to chemotherapy in three different PDX tumors as well as HT29 cells. Co-immunoprecipitation experiments showed that E2F4 forms a complex with p107 and DP-1, which also contributed to the chemotherapy resistance. Simultaneous knock-down of both E2F4 and p107 enhanced chemotherapy sensitivity more than E2F4 knock-down alone. In normal intestinal crypt cells E2F4 was dispensable for chemotherapy resistance. ChIP-seq and RNA-seq experiments revealed that LDHA is a key downstream target of E2F4. LDHA knock-down sensitizes cancer cells to chemotherapy. High LDHA expression in tumor tissues was an independent poor prognostic factor in colorectal cancer patients (n=466), as well as in non-small cell lung cancer (n=1,926) and breast cancer (n=3,554). Thus, the E2F4/p107-LDHA pathway represents a novel therapeutic target to overcome chemotherapy resistance in CSCs. Citation Format: Junichi Matsubara, Yong F. Li, Piero Dalerba, Debashis Sahoo, Taichi Isobe, Russ B. Altman, Michael F. Clarke. E2F4/p107 complex regulates chemotherapy resistance in human colorectal cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 81. doi:10.1158/1538-7445.AM2017-81

Abstract 3901: Therapeutic targeting of C-terminal binding protein: a key dependency for polyposis and cancer stem cell activity in APC mutant neoplasia

Abstract The C terminal binding proteins (CtBP) 1 and 2 are a family of transcriptional co-repressors overexpressed in a variety of cancers, and are frequently associated with poor prognosis and chemoresistance. CtBP has also been characterized in cell culture models as drivers of migration/invasion and epithelial-mesenchymal transition. CtBP mediates its transcriptional corepressor activity via its dehydrogenase domain, and inhibition of this domain interferes with CtBP oncogenic functions. The role of CtBP in APC mutant neoplasia remains obscure even though APC is responsible for degradation of both β-catenin and CtBP in suppressing colorectal tumorigenesis. Our prior work demonstrates that CtBP proteins can be effectively therapeutically targeted with substrate analogues of their intrinsic dehydrogenase domains. We now observe that pharmacologic inhibition of CtBP using either the 1st generation inhibitor 2-keto-4-methylthiobutyrate (MTOB) or the 2nd generation inhibitor hydroxyimino-3-phenylpropanoic acid (HIPP) significantly reduces the burden of intestinal polyps in the Apcmin mouse model of the human cancer predisposition syndrome Familial Adenomatous Polyposis. The number of intestinal polyps in mice with CtBP2 inhibitors were significantly lower (21 polyps/mouse for MTOB, 15 polyps/mouse for HIPP) as compared with vehicle treated mice (45 polyps/mouse) with a P value &amp;lt;0.0001. Pointing to the anti-neoplastic mechanism of MTOB and HIPP, polyps that did grow in MTOB or HIPP treated mice demonstrated robust downregulation of β catenin and Ctbp2 proteins. This observation points to the role of Ctbp2 as a putative driver oncogene that can be therapeutically targeted in vivo. In addition, CtBP2 has been reported to play an important role in human colon cancer stem cells via its interaction with the transcription factor TCF4 on chromatin. We now show that inhibiting CtBP with HIPP or HIPP analogues attenuates CtBP2 protein levels in colon cancer stem cells with resultant decreased expression of CtBP transcriptional target genes, such as the motility/invasion regulator TIAM1, and c-MYC, a known driver of the cancer stem cell phenotype. Moreover, colonospheres treated with CtBP inhibitor showed downregulation of cancer stem cell markers CD133 and c-MYC, as compared with vehicle treatment. Flow cytometry analysis of intestinal epithelium from Apcmin mice treated with CtBP inhibitor vs. vehicle indicated a significant reduction in CD133+/CXCR4+ cells that are associated with a tumor initiating cell phenotype. Our data demonstrate that CtBP inhibition may reduce colon neoplasia via downregulation of c-MYC and the cancer stem cell/tumor initiating cell compartment, and that CtBP is a promising and novel target for therapy in colon cancer. Citation Format: Ayesha T. Chawla, Agnes Cororaton, Rashmi Seth, Barbara Szomju, Evan T. Sumner, Steven R. Grossman. Therapeutic targeting of C-terminal binding protein: a key dependency for polyposis and cancer stem cell activity in APC mutant neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3901. doi:10.1158/1538-7445.AM2017-3901

Functional significance of CD105-positive cells in papillary renal cell carcinoma

BackgroundCD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties.MethodsWithin this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen (786-O, SMKT-R2, Caki-2, 796-P, ACHN, RCC6) and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 (one of the highest numbers of CD105+ cells; primary origin), ACHN (a low number of CD105+ cells; metastatic origin), HKCSC (putative positive control), and ASE-5063 (additional control).ResultsIn 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of CD105 and/or CD133 expression with the enhancement of stem-like properties.ConclusionsBased on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines.

Abstract 1714: The role of Hedgehog signaling in the regulation of human colon cancer stem cells

Abstract Recent data support a hierarchical model of colon cancer in which tumor growth is driven by a subpopulation of cancer stem cells (CSCs) that may also be the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumor would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here, as part of the OncoTrack* consortium, we report the use of Matrigel-based 3D in vitro models of patient-derived colon cancer for the characterization of molecular pathways important in the regulation of CSC self-renewal and survival. Cellular subpopulations were isolated from patient-derived 3D-culture models based on expression of the CSC marker aldehyde dehydrogenase (ALDH) and then functionally tested for tumor initiation and self-renewal capacity by limiting dilution serial xenotransplantation. These studies demonstrate CSCs to be ALDH+. ALDH+ and ALDH- cells from 3D-culture and xenograft models were then subjected to RNA sequencing for whole transcriptome analysis. These analyses demonstrated ALDH+ CSCs cells to be enriched for stem cell associated genes (ALDH1A1, LGR5, BMI1, CD44, CD166), developmental processes (embryonic, tissue development, EMT) and signaling pathways (Wnt signaling). In particular, the Hedgehog signaling pathway was found to be enriched in both 3D in vitro and xenograft models. The role of Hedgehog signaling in colon cancer remains controversial. It has been reported as a negative regulator of Wnt signaling and to be inactive in colon cancer cells lines. Here, using small molecule inhibitors and lentivirus mediated gene knockdown, we report on the role of Hedgehog signaling in the regulation of colon CSC self-renewal and identify non-canonical Hedgehog signaling as a positive regulator of Wnt signaling. *The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234. Citation Format: Joseph L. Regan, Stephanie Staudte, Dirk Schumacher, Ulrich Keilholz, Johannes Haybaeck, Hans Lehrach, Marie-Laure Yaspo, David Henderson, Andreas Steffen, Joern Toedling, Ralf Lesche, Reinhold Schaefer, Christian R. Regenbrecht, Dominik Mumberg, Martin Lange. The role of Hedgehog signaling in the regulation of human colon cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1714.

Abstract 3839: Vitamin E delta-tocotrienol targets human colon cancer stem cells and inhibits colon cancer metastasis and induces apoptosis

Abstract Background: Colon cancer is the most commonly diagnosed cancer both in men and women and cause of cancer related deaths worldwide. Treatment for the recurrent and metastatic colon cancer remains a center of clinical attention. Cancer stem cells (CSCs) are suggested to be the only cells within tumor with tumorigenic capacity and implicated in metastasis. We have shown that vitamin E delta-tocotrienol (VEDT) is the most bioactive tocotrienols against cancers. This study aimed to evaluate the effect of VEDT on colon cancer growth, metastatic epithelial to mesenchymal transition (EMT), migration/ invasion/angiogenesis and colon CSCs growth and apoptosis. Methods: Human colon cancer cells HCT-116 and metastatic colon cancer cells SW-620 and colon CSCs were treated with VEDT (10-100 μM) for 72 h and cell viability recorded. VEDT (50 μM) was used for malignant transformation; migration/invasion, CSCs spheroid formation, and pluripotency were determined. Azoxymethane treated Fisher 344 rats were used as colon carcinogenesis model. They were treated with 1) vehicle control and 2) VEDT (200 mg/kg, orally twice a day) for 20 weeks. After 20 weeks the polyps and tumor formation were recorded. Results: VEDT significantly inhibited the anchorage-dependent and independent cancer cell growth, migration/invasion and induced apoptosis compared to control. VEDT inhibited the CSC spheroid formation EMT (E-cadherin to vimentin), metastasis (MMP9), angiogenesis (VEGF), CSCs transcription factors (Nanog, Oct4, Sox2 and KLF4) compared to control. Conclusion: Vitamin E delta-tocotrienol inhibited colon cancer growth and metastasis through inhibition of cancer stem cell pluripotency, EMT, migration, invasion, angiogenesis and induction of apoptosis. Citation Format: Kazim Husain, Domenico Coppola, Said M. Sebti, Mokenge P. Malafa. Vitamin E delta-tocotrienol targets human colon cancer stem cells and inhibits colon cancer metastasis and induces apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3839.

Inhibition of c-Yes induces differentiation of HT-29 human colon cancer stem cells through midbody elongation.

Recent research suggests that a small group of cells, named cancer stem cells (CSCs), is responsible for initiating tumor formation, recurrence, and metastasis. c-Yes, a proto-oncogene that is a subfamily of Src family kinase, is often activated in human colon cancer; this implicates c-Yes in the onset and progression of the disease. The objective of this study was to investigate the correlation between c-Yes and CSCs. We performed a sphere formation assay and reverse transcription-polymerase chain reaction for studying the differentiation of HT-29 human colon CSCs. To demonstrate the specific role of c-Yes in CSCs, we performed live cell microscopy and a cell cycle assay. These study shows, for the first time, that c-Yes is enriched in CD133+ CSCs, compared to their CD133- counterparts, and that c-Yes depletion in CD133+ cells induces cell differentiation. Moreover, c-Yes depletion was found to elongate the midbody and increase the proliferation doubling time. This also suggested that the misregulation of microtubules during chromosomal separation causes aneuploidy. Our results suggest that c-Yes may play a crucial role in initiating, maintaining, and driving the tumorigenic property of colon cancer.

Establishment and Characterization of Primary Human Ovarian Cancer Stem Cell Line (CD44+ve)

Ovarian cancer is ranked as the 7th most lethal cancer worldwide with 239,000 new cases annually. The mortality rate is high because most ovarian tumors are diagnosed at advanced stages and are resistant to chemotherapy and thus incurable due to the lack of effective early detection of ovarian tumors. There is a small sub-population of ovarian tumor cells capable of self-renewal and differentiation into different cancer cell types, called cancer stem cells (CSCs), which might be responsible for cancer relapse. The CD44+ phenotype in ovarian tumor cells elucidates cancer initiating cell-like properties of promoting differentiation, metastasis, and chemotherapy-resistance. Increased expression of genes previously associated with CSCs promotes regenerative capacity by promoting stem cell function that can drive cancer relapse and metastasis. In this study we present a method to isolate the primary epithelial ovarian cancer cells from human solid tumor and establish CD44+ve primary ovarian cancer stem cell (OCSCCD44+ve) line using magnetic microbeads. Also we evaluated the expression of stemness genes Nanog, Sox2, Oct4, and Nestin by real-time qPCR analysis. Thequantitative analysis by real-time qPCRshows that OCSCCD44+ve overexpressed the embryonic stem cell marker genes Nanog, Oct4, Sox2, and Nestin when compared with ovarian cancer cells OCCCD44-ve as positive control and ovarian cells as negative control. We demonstrate that CD44 in malignant ovarian tumors is a critical molecule that exhibits cancer stem cell properties that enhance tumorigenicity and cancer metastasis. Our results provide a better understanding of ovarian CSCs, which is important for future in vivo studies with subsequent target therapy for preclinical studies.

Abstract B23: Characterization of canine glioma cancer stem cells for human glioblastoma models

Abstract Glioblastoma (GBM) is the most aggressive and common form of glioma in humans with an average survival period of twelve to fourteen months. Due to its highly invasive and malignant nature, current treatment options have not been able to improve the survival rate over the past several decades. Several studies claim that cancer stem cells (CSC), self-renewalable and differentiable into neuron- and glia-like cells, may play a key role in initiating tumor formation and cancer reoccurrence. Cancer stem cells are known to have tumorigenic ability and resistance to current cancer treatment options. Similar to humans, naturally occurring glioma in canines is highly common and devastating. Cancer in canines and humans shares strong anatomical and physiological similarities, including histological appearance, tumor genetics, molecular targets, biological behavior, and response to therapies. The goal of this study is to characterize and test the presence of canine glioma cancer stem cells (CSC) in order to utilize the spontaneously occurring cancer models to deepen understanding of human and canine GBM cancer stem cells and investigate better cancer model systems utilizing naturally occurring canine glioma since artificial rodent models have not been able to translate into human GBM studies effectively. Two different canine glioma cells lines, Roxanne and Ike, were harvested and provided from the College of Veterinary Medicine Small Animal Hospital at Purdue University. Canine cell line Roxanne, Grade II Oligodendroglioma, was harvested from a six-year-old female mixed breed by post-mortem examination. The second canine cell line Ike, Grade II Oligodendroglioma neoplastic cells with negative GFAP expression, was harvested from a six-year-old male Boxer by surgery. In order to compare these canine cell lines with the human GBM cancer stem cells GBAM1 human GBM stem cell line (known to be 98% CD133 positive) was obtained from Moffitt Cancer Center. These three cell lines were investigated by immunofluorescence with CD133, sex determining region Y-box 2 (Sox2), Notch1, glial acidic fibrillary protein (GFAP), and O6-alkylguanine DNA alkyltransferase (MGMT) protein markers. Cells were fixed and permeabilized prior to antibody attachment. Confocal images for detection of CD133, Sox2, Notch, GFAP, and MGMT were taken with a Nikon A1R-MP confocal microscope equipped with an objective Plan Fluor 20X-oil/water (0.75 NA). Roxanne and GBAM1 were tested for immunofluorescence since Ike was not able to grow confluent in in vitro environment. GBAM1 expressed all protein markers, CD133, Sox2, Notch, and MGMT, except for GFAP from the immunofluorescent detection. However, canine Roxanne only expressed Sox2 proteins, which is a transcription factor for undifferentiated stem cell. Other immunofluorescent signals were either weak or not expressed. Since the cancer stem cell population is very low in heterogeneous glioma tumor environment, it was difficult to conclude the presence of these protein markers in canine samples. In order to validate and further explore these results, western blot will be performed in the three cell lines. Characterization of both human and canine cancer stem cells has a potential to provide a better understanding of both human and canine glioma cancer stem cells, improve an animal cancer model system, and develop more effective treatments for glioblastoma. Citation Format: Soo Jung Ha, Marisol Herrera-Perez, Jiri Adamec, R. Timothy Bentley, Jenna L. Rickus, Kari L. Clase. Characterization of canine glioma cancer stem cells for human glioblastoma models. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B23.