Tumor-initiating Cells Function Research Articles

Abstract PO5-24-02: Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer

Abstract Background: A subpopulation of tumor cells known to have intrinsic resistance to therapies and contribute to metastasis function as “cancer stem”, or tumor-initiating, cells (TICs). TICs can be identified by various biomarkers, including fluorescent reporters for signaling pathways that regulate TIC function such as STAT signaling. However, standard TIC reporters have limitations. Their expression is unstable and there is no established method to follow TICs as they undergo cell state changes. Furthermore, we do not completely understand the transcriptional features of TICs. Improved methods of identifying TICs as well as the development of high-resolution transcriptional signatures are essential to understand TIC biology. An improved understanding of TIC biology can inform the development of novel therapeutics to selectively ablate the TIC compartment, thus improving response to standard cancer therapies. Methods: We screened a panel of PDX models to determine whether a STAT reporter identifies TICs in patient-derived xenograft (PDX) models. To augment existing STAT TIC reporters, we developed a two-component STAT signaling-specific lineage-tracing (LT) system. The first component labels active STAT signaling cells by expression of enhanced green fluorescent protein (EGFP+), followed by a self-cleaving P2A peptide and a TAM-inducible Cre-recombinase (4M67-EGFP-P2A-CreERT2). The second component is a constitutively expressed dual-color switching Cre-dependent reporter vector (EFS-loxPdsRedloxP-mNeptune2). We determined which LT cell populations (EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP−/mNeptune2+, and EGFP−/dsRed+) from SUM159 xenografts functioned as TICs. To probe TIC transcriptional features, we performed scRNA seq on the two tumor models with STAT TICs and a PDX model that did not have STAT TICs. We performed lineage trajectory analysis and differential gene expression analysis to characterize candidate TIC transcriptional signatures. To confirm whether these transcriptional features are associated with TICs, we identified a biomarker of this cell state (bone marrow stromal antigen 2 [BST2]) and explored the relationship between this biomarker and TICs. Results: We identified four PDX models with STAT reporter activity, one of which had STAT TICs. We validated our LT system in several models both in vitro and in vivo, then demonstrated EGFP+/mNeptune2+ from SUM159 xenografts are enriched for TICs and that lineage-tagged cells (mNeptune2+) function as proliferative early progenitor cells. scRNA seq of three xenograft models uncovered a distinct antiviral cell state in all three models that represent a more highly enriched TIC subpopulation. Critically, transcriptional features were shared between the antiviral cell states across xenograft models. To determine whether cells in this antiviral state represent TICs, we FACS-enriched BST2 cells (an IFN-stimulated cell surface protein that was a marker of the antiviral cell state) and demonstrated enrichment of the TIC population by mammosphere formation assays and limiting dilution transplantation. We demonstrated that BST2 expression is increased in residual PDX tumors following chemotherapy. Furthermore, we demonstrated similar antiviral cell states are present in human breast cancer scRNA seq datasets. Conclusions: These data demonstrate TICs adopt an antiviral cell state in some triple-negative breast cancers, and BST2 is a function marker of TICs in this cell state. Therefore, targeting genes associated with this cell state or associated antiviral pathways may represent a therapeutic vulnerability to eliminate TICs in some breast cancers. Citation Format: Eric Souto, Ping Gong, John Landua, Ram Srinivasan, Lacey Dobrolecki, Abhinaya Ganesan, Michael Lewis. Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-02.

Cancer-associated fibroblasts nurture LGR5 marked liver tumor-initiating cells and promote their tumor formation, growth, and metastasis.

In liver cancer, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) compartment represents an important tumor-initiating cell (TIC) population and served as a potential therapeutic target. Cancer-associated fibroblasts (CAFs) is a critical part of the tumor microenvironment, heavily influenced TIC function and fate. However, deeply investigations have been hindered by the lack of accurate preclinical models to investigate the interaction between CAFs and TIC. Organoids model have achieved major advancements as a precious research model for recapitulating the morphological aspects of organs, and thus also serving as a candidate model to investigate the mutual interaction between different cell types. Consequently, this study aimed to construct a three-dimensional (3D) co-culture organoid model of primary LGR5-expressing tumor stem cells from primary murine liver tumors with CAFs to investigate the impact of CAFs on LGR5 marked TICs in liver cancer. First, both of the transgenic LGR5-diphtheria toxin receptor (DTR)-GFP knock-in mice and transgenic Rosa26-mT mice developed primary liver tumors by diethylnitrosamine (DEN) administration. Tumor organoids and CAFs were generated from those primary liver cancer separately. Second, LGR5-expressing TICs organoid with CAFs were established ex vivo based on cell-cell contact or trans-well co-culture system, and the mutual influence between those two types of cells was further investigated. Subsequently, immunodeficient mouse-based xenograft model was further adopted to evaluate the influence of CAFs to LGR5 tumor stem cell, tumor formation, and metastasis. The co-culture organoid model composed of murine liver tumor LGR5+ tumor-initiating cells and CAFs in 3D co-culture was successfully established, with the intention to investigate their mutual interaction. The existence of CAFs upon engrafting tumor organoids resulted in dramatic higher number of LGR5+ cells in the neoplasia when compared with engrafting tumor organoids alone. Furthermore, ex vivo culture of isolated LGR5+ cells from tumors of co-engrafted mice formed significantly larger size of organoids than mono-engrafted. Our results also indicated significantly larger size and number of formed organoids, when LGR5+ cells co-cultured with CAF in both cell-cell contact and paracrine signaling in vitro, comparing to LGR5+ cells alone. Furthermore, we found that specific knockout of LGR5 expressing cells suppressed CAF-mediated promotion of tumor formation, growth, and metastasis in the experimental mice model. Altogether, in a 3D co-culture type of murine liver LGR5+ cells and cancer-associated fibroblasts, we have demonstrated robust effects of CAFs in the promotion of LGR5 marked liver TICs. We also further revealed the influence of tumor microenvironment on stem cell-related therapy, suggesting the possibility of combing CAF-targeted and tumor stem cell targeted therapy in treating liver cancer.

Abstract P6-12-04: An Inducible Lineage Tracing System for the Identification of Tumor-Initiating Cells

Abstract Background: Metastasis and therapeutic resistance are a major clinical challenge, responsible for the vast majority of cancer deaths. A subpopulation of tumor cells known to have intrinsic resistance to standard therapies and contribute to metastasis function as “cancer stem”, or tumor-initiating, cells (TICs). Enriched populations of TICs are typically identified by markers such as aldehyde dehydrogenase activity, the cell surface marker combination of CD44+/CD24-, or fluorescent reporters for signaling pathways that regulate TIC function such as STAT3. Despite their utility, TIC markers and reporters have limitations. Marker expression can be unstable and there is no established method to lineage trace long-lived TICs or to follow them as they undergo cell state changes. Methods: To augment existing TIC reporters, we developed a two component TAM-inducible, Cre recombinase-dependent, STAT3 signaling-specific lentiviral LT system. The first component is a vector that labels cells with active STAT3 signaling (EGFP+), followed by a self-cleaving peptide and TAM-inducible Cre-recombinase (4M67-EGFP-P2A-CreERT2). The second component is a constitutively expressed dual-color switching Cre-dependent reporter vector (EFS-loxPdsRedloxP-mNeptune2). Addition of TAM drives color switching from dsRed to mNeptune2 via CreERT2 recombination in STAT3 signaling cells. Both lentiviral vectors were constructed using Gateway® Cloning. Sum159 cells were transduced with the LT system and reporter activity was validated both in vitro and in vivo using confocal microscopy and flow cytometry. Four LT cell populations (EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP-/mNeptune2+, and EGFP-/dsRed+) were enriched using fluorescence activated cell sorting, then analyzed by single cell RNA sequencing (scRNA seq). Results: Our results confirm the STAT3 LT reporter identifies STAT3 signaling cells (EGFP+), which can be labelled (mNeptune2+) upon addition of TAM. We conducted a TAM dose response curve to identify the optimal TAM dose for complete and partial labeling of STAT3 signaling cells. scRNA seq uncovered gene expression patterns within the TIC compartment and revealed similarities and differences in gene expression between the TIC compartment and the remaining LT reporter populations. Conclusion: These data demonstrate our LT system is a powerful tool that can be applied to uncover the role of TICs in metastasis and therapeutic resistance, as well as identify genetic vulnerabilities to specifically target TICs. Citation Format: Eric P. Souto, Michael T. Lewis. An Inducible Lineage Tracing System for the Identification of Tumor-Initiating Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-04.

Abstract 1689: Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts

Abstract Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, WNT, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents. Citation Format: John Landua, Ping Gong, Michael T. Lewis. Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts [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 1689.

OS06.3A LSD1-directed therapy curtails glioblastoma tumorigenicity by limiting the adaptation of tumor initiating cells to stressful environments

Abstract BACKGROUND Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, therapy resistance and poor blood-brain-barrier penetration hinder the amelioration of the standard-of-care. Included in the GBM mass are the tumor initiating cells (TICs), representing the driver of GBM growth and relapse in virtue of their stem-like traits and therapy-resistance. Being constantly exposed to environmental stress, including nutrients deficiency, hypoxia and therapeutic insults, all GBM cells -and TICs in particular- have to be highly adaptive in order to survive. Thus, their ability to cope with stress could be targeted to curtail TICs maintenance and the whole GBM aggressiveness. The key of TICs adaptation relies, among the others, on their epigenetic plasticity, hence encouraging epigenetic drugs testing. MATERIAL AND METHODS By exploiting patient-derived GBM TICs and orthotopic xenograft models, we tested the antitumorigenic features of a novel, selective, orally bioavailable and brain-penetrant Lysine-specific histone demethylase 1 inhibitor (LSD1i). We confirmed the specificity of its effects by LSD1 genetic targeting. A combination of RNA-seq, Chromatin Immunoprecipitation(ChIP)-seq, Mass Spectrometry and reverse genetic experiments unraveled LSD1 molecular players in GBM TICs. RESULTS We identified LSD1 as a druggable target in human GBM: LSD1i treatment, mirrored by LSD1 genetic targeting, impairs growth, viability, stem-like traits and in vivo tumorigenicity of GBM TICs. Mechanistically, LSD1 is crucial for the expression of the activating transcription factor 4 (ATF4), which coordinates the integrated stress response (ISR) to manage stressful stimuli as nutrient deprivation and endoplasmic reticulum stress. By mimicking these stress cues in vitro, we found that LSD1i triggers a delayed but unabated ATF4 translation which provokes an over-lasting ISR, eventually culminating in GBM TICs apoptosis. Lastly, LSD1 demethylase activity is dispensable for ATF4 induction. Rather, LSD1i exerts its anti-tumorigenic potential by interfering with LSD1 scaffolding function in GBM TICs. CONCLUSION LSD1-directed therapy is likely a promising strategy to hinder GBM. By sensitizing GBM TICs to stress, LSD1i endangers the GBM TICs pool. The effectiveness of LSD1i administration in different patient-derived GBM TICs and xenografts, regardless of their molecular profile, places a strong rationale toward the clinical translation of this approach for GBM management. FUNDING Italian association for Cancer Research (AIRC) and Italian Ministry of Health

MicroRNA-454 promotes liver tumor-initiating cell expansion by regulating SOCS6

Tumor-initiating cells (T-ICs) are involved in the tumorigenesis, progression, drug resistance and recurrence of hepatocellular carcinoma (HCC). However, the underlying mechanism for the propagation of liver T-ICs remains unclear. Herein, we find that miR-454 is upregulated in liver T-ICs and has an important function in liver T-ICs. Functional studies have revealed that knockdown of miR-454 inhibits liver T-IC self-renewal and tumorigenesis. Conversely, forced miR-454 expression promotes liver T-IC self-renewal and tumorigenesis. Mechanistically, we found that miR-454 downregulates SOCS6 expression in liver T-ICs. The correlation between miR-454 and SOCS6 is validated in human HCC tissues. Furthermore, HCC cells that overexpress miR-454 are resistant to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrates that miR-454 may predict sorafenib benefits in HCC patients. In conclusion, our findings reveal the crucial role of miR-454 in liver T-IC expansion and sorafenib response.

Abstract P4-06-10: Lentiviral signaling reporters uncover tumor-initiating cell heterogeneity in breast cancer patient-derived xenografts

Abstract Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents. Citation Format: John Landua, Ping Gong, Michael Lewis. Lentiviral signaling reporters uncover tumor-initiating cell heterogeneity in breast cancer patient-derived xenografts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-10.

MicroRNA-29a regulates liver tumor-initiating cells expansion via Bcl-2 pathway

MicroRNAs (miRNAs) participate in tumorigenesis, progression, recurrence and drug resistance of hepatocellular carcinoma (HCC). However, few miRNAs have been identified and entered clinical practice. Herein, we report that miR-29a is downregulated in tumor-initiating cells (T-ICs) and has an important function in liver T-ICs. Functional studies revealed that miR-29a knockdown promotes liver T-ICs self-renewal and tumorigenesis. Conversely, a forced miR-29a expression inhibits liver T-ICs self-renewal and tumorigenesis. Mechanistically, we find that miR-29a downregulates Bcl-2 via binding its mRNA 3′UTR in liver T-ICs. The correlation between miR-29a and Bcl-2 is validated in human HCC tissues. Furthermore, the miR-29a expression determines the responses of hepatoma cells to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrated that the miR-29a high patients are more sensitive to sorafenib treatment. In conclusion, our findings revealed the crucial role of the miR-29a in liver T-ICs expansion and sorafenib response, rendering miR-29a as an optimal target for the prevention and intervention of HCC.

EP1.14-14 MUC1 Confers Chemotherapy Resistance of Tumor-Initiating Cells Through EGFR-IL-6 Axis

Tumor initiating cells (TICs) are responsible for causing chemotherapy resistance and tumor relapse, but the mechanisms have not been elucidated. This study aims to explore the role of mucin 1 (MUC1) in chemotherapy resistance of TICs and its clinical significance. The relationships between expression of MUC1 and clinical outcomes were analyzed by multiple databases. The MUC1 expression and function in chemoresistant TICs were investigated in vitro and in vivo through gain-of and loss-of function strategies. The potential downstream targets of MUC1 were analyzed using chromatin immunoprecipitation and luciferase reporter assay. The prognostic value of MUC1 signaling was evaluated using IHC. High expression of MUC1 positively related with poor outcome of cancer patients. Elevation of MUC1 in chemoresistant cancer cells concomitant with TICs enrichment. Mechanistically, MUC1 induces the expansion of TICs population through activation of EGFR, which augments interleukin-6 (IL-6) transcription. Inhibition of EGFR-IL-6 signaling significantly abolishes expansion of chemoresistant TICs. Furthermore, coadministration of EGFR inhibitor overcomes the resistance of TICs to paclitaxel both in vitro and in vivo. Analysis of cervical cancer patients reveales that the expression of MUC1, EGFR and IL-6 are considerably increased after chemotherapy, showing a positive correlation between the expression of MUC1, EGFR and IL-6. Consistently, mining TCGA datasets uncovered a significant positive correlation between activation of MUC1-EGFR-IL-6 signaling pathway and poor disease-free survival in chemo-treated cervical cancer patients. MUC1 confers chemotherapy resistance of TICs through EGFR-mediated transcriptional regulation of IL-6.

Tumor-intrinsic PD-L1 reduces actin cytoskeleton polymerization to promote mTORC1 signals driving tumor stemness

Abstract Tumor-intrinsic PD-L1 signals contribute to tumor virulence and pathology, but mechanisms are poorly understood. We previously showed that tumor-intrinsic PD-L1 increases mTORC1 and tumor initiating cell (TIC) generation and function in ovarian cancer and melanoma. We used CRISPR/Cas9 (KO) and shRNA (lo) to reduce PD-L1 in mouse melanoma (B16) and ovarian cancer (ID8agg), and human ovarian cancer (ES2). PD-L1lo reduced mTORC1 and TIC as expected, but unexpectedly increased actin polymerization and filopodia, and altered cell morphology. The actin depolymerizing agents Latrunculin-A or Cytochalasin-D reversed actin polymerization in PD-L1lo ID8agg cells and increased mTORC1 (p-rpS6) without affecting mTORC2 (pAktS473). Latrunculin-A and Cytochalasin-D increased TIC numbers and stemness gene expression (Oct4) in PD-L1lo ID8agg cells with no effect on control ID8agg cells. Similar actin effects were seen in PD-L1KO B16 and PD-L1KO ES2. To confirm TIC function, Latrunculin-A increased tumorospheres (TIC self-renewal function) in PD-L1lo, but not control ID8agg cells. The mTORC1 inhibitor rapamycin reduced TIC numbers and functions with no effect on actin polymerization, suggesting mTORC1 is downstream of actin polymerization. Rptorlo ID8agg cells (with low mTORC1 signaling) did not exhibit the PD-L1lo actin polymerization, nor did Latrunculin-A increase Rptorlo ID8agg TIC numbers, tumorospheres, or stemness genes, confirming the flow of signals is PD-L1 to actin to mTORC1 to TIC (stemness). Our data define a highly novel tumor virulence control mechanism of cell-intrinsic PD-L1 through inhibiting actin polymerization suggesting new drug discovery targets. Preliminary data show some similar effects of tumor PD-1.

Abstract 4177: Anticancer agent Aminoflavone restores the expression of tumor suppressor miRNA 26a and inhibits putative stemness biomarker α6-integrin in Tamoxifen resistant cells

Abstract Despite the efficacy of anti-estrogen agent Tamoxifen, commonly used to treat patients with estrogen receptor positive (ER+) tumors, up to 40% of patients experience recurrence. Breast tumor-initiating cells (TICs), or breast cancer stem cells, exhibit Tamoxifen resistance and contribute substantially to recurrence. We recently demonstrated that investigational anticancer agent Aminoflavone (AF) disrupts mammospheres (in vitro clusters of cells enriched with TICs) by thwarting the expression of α6-integrin. In the current study, we found AF potently inhibited Tamoxifen resistant (TamR) cell growth and blocked Tamoxifen-mediated stimulation of TamR cell proliferation using the Alamar Blue assay. qPCR analyses revealed α6-integrin expression was significantly elevated in ER+ breast cancer cell models of acquired and de novo Tamoxifen resistance relative to Tamoxifen sensitive cells. In particular, AF decreased the expression of both A and B variants of α6-integrin, the B variant being essential for TIC function. Western blotting revealed AF reduced total α6-integrin expression in TamR cells. Furthermore, we found that an anti-α6-integrin blocking antibody sensitized TamR cells to the active Tamoxifen metabolite, 4-hydroxy-Tamoxifen, and enhanced the efficacy of AF in these cells. AF also reduced the protein expression of p-Src, a downstream target of α6-integrin that is linked to Tamoxifen resistance and decreased breast cancer survival. In addition, miRNA sequencing of Tamoxifen sensitive and TamR mammospheres revealed differential expression of several miRNAs. Notably, miR-26a expression was down-regulated 2-fold in TamR mammospheres compared to Tamoxifen sensitive mammospheres and AF restored miR-26a expression 5-fold in TamR mammospheres. Using miRNA target prediction algorithms TargetScan and PicTar, we found miR-26a binding sites on the α6-integrin promoter. Taken together, our data suggest that AF re-expresses tumor suppressor miR-26a and inhibits the α6-integrin/Src signaling axis to reduce TIC capacity and counteract Tamoxifen resistance. Citation Format: Petreena Campbell, Leah Rowland, Anna Opoku-Agyeman, Nichole Mavingire, Ubaldo Soto, Gayathri Nagaraj, Yonghong Zhang, Sean (Xin) Chen, Charles Wang, Eileen Brantley. Anticancer agent Aminoflavone restores the expression of tumor suppressor miRNA 26a and inhibits putative stemness biomarker α6-integrin in Tamoxifen resistant 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 4177. doi:10.1158/1538-7445.AM2017-4177

Abstract 2003: C-terminal binding protein (CtBP): An emerging oncogene and small molecule drug target in solid tumors

Abstract C-terminal binding proteins 1 and 2 (CtBP) are transcriptional coregulators whose overexpression has been linked to poor prognosis and/or chemoresistance in most common solid tumor types. CtBP exerts oncogenic activities through modulation of gene expression programs governing cell survival, epithelial/mesenchymal transitions, migration/invasion, and cell cycle, and is also required for colon cancer tumor initiating cell function. CtBP, however, has never been formally characterized as an oncogene. We now show that Ctbp2 exhibits transforming activity in immortalized NIH 3T3 as well as mouse and human primary cells. Ctbp2 alone, or in cooperation with SV40 large T antigen (LT) transformed NIH 3T3 cells and MEF’s, respectively, to anchorage independence with similar efficiency to mutant H-Ras. Human BJ foreskin fibroblasts were also transformed to anchorage independence by CtBP2 in cooperation with LT, SV40 small T antigen, and h-TERT with efficiency similar to H-Ras, indicating that CtBP overexpression, as found in the majority of human colon, ovary, breast, and prostate cancers, may be a key oncogenic driver gene. To confirm the physiologic role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 hemizygote (Ctbp2+/-) mice, which are otherwise healthy. CtBP is a known target of the APC E3 ligase and is thus stabilized in APC mutated human colon cancers and is found in high levels in APCmin polyps. Remarkably, survival to humane endpoint at 37 weeks for Apcmin/+ vs. Apcmin/+-Ctbp2+/- mice was 0% vs. 100% (p <0.001). As CtBP also encodes a targetable intrinsic dehydrogenase that forms an NADH-dependent oligomerization surface, our cell and mouse model data supports the idea that CtBP could be an attractive small molecule drug target in tumors where it is upregulated. We have therefore synthesized a series of dehydrogenase substrate competitive inhibitor compounds based on CtBP's natural substrate 4-methylthio-2-oxobutyric acid (MTOB) that utilize a phenylpyruvate backbone and demonstrate nanomolar enzymatic IC50's with micromolar cellular GI50’s, while exhibiting minimal off target inhibition of lactate dehydrogenase. Phenylpyruvate—based CtBP inhibitors biochemically caused dissolution of CtBP oligomers, which are the active transcriptional conformation, and exhibited on-target transcriptional effects in cells, with reversal of CtBP transcriptional repression of key cancer target genes, such as E-cadherin, Bik, and BRCA1. In summary, we have characterized CtBP2 as a cellular proto-oncogene that can transform primary mouse and human cells in a manner similar to activated H-Ras, genetically validated Ctbp2 as a therapeutic target in the Apcmin/+ mouse model, and identify promising small molecule lead inhibitors for future therapeutic development. Citation Format: Evan T. Sumner, Sudha Korwar, Benjamin L. Morris, Martin M. Dcona, Brendan J. Hilbert, William E. Royer, Keith C. Ellis, Steven Grossman. C-terminal binding protein (CtBP): An emerging oncogene and small molecule drug target in solid tumors. [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 2003.

Chromosomal Instability Affects the Tumorigenicity of Glioblastoma Tumor-Initiating Cells.

Tumors are dynamic organs that evolve during disease progression with genetic, epigenetic, and environmental differences among tumor cells serving as the foundation for selection and evolution in tumors. Tumor-initiating cells (TIC) that are responsible for tumorigenesis are a source of functional cellular heterogeneity, whereas chromosomal instability (CIN) is a source of karyotypic genetic diversity. However, the extent that CIN contributes to TIC genetic diversity and its relationship to TIC function remains unclear. Here, we demonstrate that glioblastoma TICs display CIN with lagging chromosomes at anaphase and extensive nonclonal chromosome copy-number variations. Elevating the basal chromosome missegregation rate in TICs decreases both proliferation and the stem-like phenotype of TICs in vitro Consequently, tumor formation is abolished in an orthotopic mouse model. These results demonstrate that TICs generate genetic heterogeneity within tumors, but that TIC function is impaired if the rate of genetic change is elevated above a tolerable threshold. Genetic heterogeneity among TICs may produce advantageous karyotypes that lead to therapy resistance and relapse; however, we found that TICs have an upper tolerable limit for CIN. Thus, increasing the chromosome missegregation rate offers a new therapeutic strategy to eliminate TICs from tumors. Cancer Discov; 6(5); 532-45. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 461.

Abstract LB-077: Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer

Abstract The Wnt signaling pathway, which controls TCF/Lef/β-catenin (TCF) transcription, is well accepted as a dominate player in CRC tumor initiation and progression. More recently, this pathway has been implicated as a major driving force inducing epithelial-mesenchymal transition (EMT), acquiring a drug resistant tumor initiating cell (TIC) phenotype that may promote metastasis. As a result, this pathway has been seriously pursued for novel drug development that has yielded no real translational success due to the lack of “druggable” targets. Thus, the major challenge moving forward is to identify a druggable target within the Wnt/TCF pathway that may result in effective clinical translation. Using a chemical biology approach targeting EMT we have identified topoisomerase IIα (TopoIIα) as a master regulator EMT by participating as a required component of TCF-transcription in CRC. Specifically, we show that inhibiting TopoIIα using small molecule ATP-competitive inhibitors as well as shRNA results in the blockade of TCF-transcription using the TOP/FOP flash reporter system. Inhibition of TopoIIα-dependent TCF-transcription results in significant reversion of EMT characterized by Western blot analysis in a panel of CRC cell lines. Time course studies reveal that inhibiting TopoIIα results in downregulation of TCF-transcription followed by the induction of apoptosis using 3D multicellular tumor spheroid models. We show that TopoIIα participates in protein-protein complexation with β-catenin using co-immunoprecipitation studies, which is not prevented by small molecule inhibitors. However, chromatin immunoprecipitation (ChIP) studies reveal that ATP-competitive TopoIIα inhibitors can significantly block TCF-transcription by preventing TCF-complex-DNA binding. In addition, inhibiting TopoIIα-dependent TCF-transcription correlates with the inhibition of TIC function characterized by loss of colony formation using the clonogenic assay. Likewise, inhibiting TopoIIα-dependent TCF-transcription inhibits the invasive potential in a panel of CRC tumor cell lines. Interestingly, the clinically used TopoIIα drug, etoposide, had no effect on TCF-transcription, EMT, or invasion. In conclusion, we hypothesize that TopoIIα participates as a component of the TCF-complex via the C-terminus and is required for binding chromatin DNA and initiating gene transcription. TopoIIα participation in TCF-transcription may convey drug resistance due to altered poison binding sites. However, our data indicates that TopoIIα-dependent TCF-transcription requires ATP. Hence, central to our hypothesis is that N-terminal ATP sites are conserved providing an Achilles heel to drug resistance. Therefore, N-terminal ATP binding sites provide optimal druggable target sites that may be utilized for translational drug development preventing TCF-transcription, EMT and potentially metastasis in CRC patients. Citation Format: Daniel V. LaBarbera, Qiong Zhou, Adedoyin D. Abraham, Linfeng Li, Wells A. Messersmith. Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-077. doi:10.1158/1538-7445.AM2015-LB-077

Identifying and targeting tumor-initiating cells in the treatment of breast cancer.

Breast cancer is the most common cancer in women (excluding skin cancer), and it is the second leading cause of cancer-related deaths. Although conventional and targeted therapies have improved survival rates, there are still considerable challenges in treating breast cancer, including treatment resistance, disease recurrence, and metastasis. Treatment resistance can be either de novo - because of traits that tumor cells possess before treatment - or acquired - because of traits that tumor cells gain in response to treatment. A recently proposed mechanism of de novo resistance invokes the existence of a specialized subset of cancer cells defined as tumor-initiating cells (TICs), or cancer stem cells (CSCs). TICs have the capacity to self-renew and to generate new tumors that consist entirely of clonally derived cell types present in the parental tumor. There are data to suggest that TICs are resistant to many conventional cancer therapies and that they can survive treatment in spite of dramatic shrinkage of the tumor. Residual TICs can then eventually regrow, which results in disease relapse. It has also been hypothesized that TIC may be responsible for metastatic disease. If these hypotheses are correct, targeting TICs may be imperative for achieving a cure. In the present review, we discuss evidence for breast TICs and their apparent resistance to conventional chemotherapy and radiotherapy as well as to various targeted therapies. We also address the potential impact of breast TIC plasticity and metastatic potential on therapeutic strategies. Finally, we describe several genes and signaling pathways that appear to be important for TIC function and may represent promising therapeutic targets.

STAT3 signaling is activated preferentially in tumor-initiating cells in claudin-low models of human breast cancer.

In breast cancer, a subset of tumor-initiating cells (TIC) or "cancer stem cells" are thought to be responsible for tumor maintenance, treatment resistance, and disease recurrence. While current breast cancer stem cell markers (e.g., CD44(high) /CD24(low/neg) , ALDH positive) have allowed enrichment for such cells, they are not universally expressed and may actually identify distinct TIC subpopulations in the same tumor. Thus, additional markers of functional stem cells are needed. The STAT3 pathway is a critical regulator of the function of normal stem cells, and evidence is accumulating for its important role in breast cancer stem cells. However, due to the lack of a method for separating live cells based on their level of STAT3 activity, it remains unknown whether STAT3 functions in the cancer stem cells themselves, or in surrounding niche cells, or in both. To approach this question, we constructed a series of lentiviral fluorescent (enhanced green fluorescent protein, EGFP) reporters that enabled flow cytometric enrichment of cells differing in STAT3-mediated transcriptional activity, as well as in vivo/in situ localization of STAT3 responsive cells. Using in vivo claudin-low cell line xenograft models of human breast cancer, we found that STAT3 signaling reporter activity (EGFP(+) ) is associated with a subpopulation of cancer cells enriched for mammosphere-forming efficiency, as well as TIC function in limiting dilution transplantation assays compared to negative or unsorted populations. Our results support STAT3 signaling activity as another functional marker for human breast cancer stem cells thus making it an attractive therapeutic target for stem-cell-directed therapy in some breast cancer subtypes.

Small nucleolar RNA signatures of lung tumor-initiating cells.

BackgroundNon-small cell lung cancer (NSCLC) is the number one cancer killer. Tumor-initiating cells (TICs) are responsible for tumor progression and recurrence. Emerging evidences suggest that small nucleolar RNAs (snoRNAs) play malfunctioning roles in lung tumorigenesis. This study aims to determine if snoRNAs have important function in lung TICs by: 1) profiling and comparing snoRNA expression patterns in lung ALDH1+/- cells of 28 primary NSCLC tissues to identify new signatures of TICs; 2) determining prognostic significance of the snoRNA signatures by analyzing the expression in 82 NSCLC tissues with different stages and histological types using quantitative PCR; 3) functionally investigating if the snoRNAs contribute to stemness of lung TICs using in vitro and in vivo assays.ResultsTwenty-two snoRNAs were identified whose changes were specific to the TICs. The expression of two snoRNAs (snoRA3 and snoRA42) was inversely associated with survival of NSCLC patients (P = 0.002, p = 0.001, respectively). Functional analysis indicated that snoRA42 was upregulated in CD133+ cells isolated from NSCLC cell lines compared with the CD133- counterparts. snoRA42 knockdown reduced the proliferation and self-renewal of TICs in vitro. However, ectopic expression of snoRA42 in non-TICs enhanced the potentials of cell proliferation and self-renewal. snoRA42 expression was associated with expression of stem cell-core transcription factors in lung TICs. Blocking snoRA42 expression in TIC xenografts decreased tumorigenesis in mice.ConclusionsThe snoRNA signatures of lung TICs provide potential biomarkers for predicting outcome of NSCLC. snoRA42 is one of the important snoRNAs in regulating features of lung TICs, and thus contributes to lung tumorigenesis.

Cool‐1‐Mediated Inhibition of c‐Cbl Modulates Multiple Critical Properties of Glioblastomas, Including the Ability to Generate Tumors In Vivo

We discovered that glioblastoma (GBM) cells use Cool-1/β-pix to inhibit normal activation of the c-Cbl ubiquitin ligase via the redox/Fyn/c-Cbl pathway and that c-Cbl inhibition is critical for GBM cell function. Restoring normal c-Cbl activity by Cool-1 knockdown in vitro reduced GBM cell division, almost eliminated generation of adhesion-independent spheroids, reduced the representation of cells expressing antigens thought to identify tumor initiating cells (TICs), reduced levels of several proteins of critical importance in TIC function (such as Notch-1 and Sox2), and increased sensitivity to BCNU (carmustine) and temozolomide (TMZ). In vivo, Cool-1 knockdown greatly suppressed the ability of GBM cells to generate tumors, an outcome that was c-Cbl dependent. In contrast, Cool-1 knockdown did not reduce division or increase BCNU or TMZ sensitivity in primary glial progenitor cells and Cool-1/c-Cbl complexes were not found in normal brain tissue. Our studies provide the first evidence that Cool-1 may be critical in the biology of human tumors, that suppression of c-Cbl by Cool-1 may be critical for generation of at least a subset of GBMs and offer a novel target that appears to be selectively necessary for TIC function and modulates chemoresistance in GBM cells. Targeting such proteins that inhibit c-Cbl offers potentially attractive opportunities for therapeutic development.

Stromal Contributions to Radiation Resistance of Breast Cancer Stem Cells

Breast cancer that is refractory to radiation therapy causes significant morbidity and mortality. Tumor-initiating cells (TICs), also called cancer stem cells (CSCs), display a radioresistant phenotype. Normal stem cells depend on stromal cells for the formation of a stem cell “niche”, but the role of the stromal cells in breast TIC function and treatment resistance is not well understood. We sought to define the effects of stromal fibroblasts on breast TIC self-renewal and resistance to IR (ionizing radiation). For investigations into breast cancer TIC and stromal fibroblast physiology we utilized MMTV-Wnt-1 transgenic mice, which spontaneously generate mammary tumors. These tumors can be dissociated into single cell suspensions and subjected to fluorescence-activated cell sorting (FACS) for in vitro colony assays or molecular analyses. The TIC population in this model has been previously identified based on cell surface markers and has been validated using in vivo limiting dilution analyses. Using this model system, we isolated normal and tumor-associated fibroblasts from primary normal and tumor murine breast tissues by FACS, and their effects on TICs were assayed in a 3D co-culture system. We show that co-culture with primary stromal fibroblasts promotes the expansion of mouse mammary TICs in vitro and maintains their ability to form new tumors in vivo. At clinically relevant doses of IR, DNA double strand breaks are mitigated and clonogenic growth of TICs is enhanced by co-culture with the stromal fibroblasts. Consistent with these observations, the TICs that are grown in co-culture maintain a more basal differentiation phenotype and harbor lower levels of reactive oxygen species (ROS) than TICs alone. The reduction in ROS found in the co-cultured TICs is a consequence of fewer mitochondria and less active oxidative phosphorylation in these cells. Working with primary tissues, we have demonstrated that stromal fibroblasts from mammary tumors play an important role in promoting TIC proliferation and resistance to IR. Our work has led to a better characterization of the unique microenvironment within the TIC-stromal niche. Future endeavors into pharmacological disruption of the supportive cues within the niche could lead to novel radiosensitizers and CSC-directed therapies.

Intestinal Tumorigenesis Initiated by Dedifferentiation and Acquisition of Stem-Cell-like Properties

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.