Subpopulation Of Tumor-initiating Cells 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.

Calcium Channel α2δ1 is Essential for Pancreatic Tumor-Initiating Cells through Sequential Phosphorylation of PKM2

Tumor-initiating cells (TICs) drive pancreatic cancer tumorigenesis, therapeutic resistance, and metastasis. However, TICs are highly plastic and heterogenous, which impede the robust identification and targeted therapy of such a population. The aim of this study is to identify the surface marker and therapeutic target for pancreatic TICs. We isolated voltage-gated calcium channel α2δ1 subunit (isoform 5)-positive subpopulation from pancreatic cancer cell lines and freshly resected primary tissues by fluorescence-activated cell sorting and evaluated their TIC properties by spheroid formation and tumorigenic assays. Coimmunoprecipitation was used to identify the direct substrate of CaMKⅡδ. We demonstrate that the voltage-gated calcium channel α2δ1 subunit (isoform 5) marks a subpopulation of pancreatic TICs with the highest TIC frequency among the known pancreatic TIC markers tested. Furthermore, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca2+ influx, which activates CaMKⅡδ to directly phosphorylate PKM2 at T454 that results in subsequent phosphorylation at Y105 to translocate into nucleus, enhancing the stem-like properties. Interestingly, blocking α2δ1 with its specific antibody has remarkably therapeutic effects on pancreatic cancer xenografts by reducing TICs. α2δ1 promotes pancreatic TIC properties through sequential phosphorylation of PKM2 mediated by CaMKⅡδ, and targeting α2δ1 provides a therapeutic strategy against TICs for pancreatic cancer.

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.

Calcium channel \u03b12\u03b41 subunit is a functional marker and therapeutic target for tumor-initiating cells in non-small cell lung cancer

It is hypothesized that tumor-initiating cells (TICs) with stem cell-like properties constitute a sustaining force to drive tumor growth and renew fully established malignancy. However, the identification of such a population in non-small cell lung carcinoma (NSCLC) has been hindered by the lacking of reliable surface markers, and very few of the currently available surface markers are of functional significance. Here, we demonstrate that a subpopulation of TICs could be specifically defined by the voltage-gated calcium channel α2δ1 subunit from non-small cell lung carcinoma (NSCLC) cell lines and clinical specimens. The α2δ1+ NSCLC TICs are refractory to conventional chemotherapy, and own stem cell-like properties such as self-renewal, and the ability to generate heterogeneous tumors in NOD/SCID mice. Moreover, α2δ1+ NSCLC cells are more enriched for TICs than CD133+, or CD166+ cells. Interestingly, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca2+ influx into cells, which subsequently activate Calcineurin/NFATc2 signaling that directly activates the expression of NOTCH3, ABCG2. Importantly, a specific antibody against α2δ1 has remarkably therapeutic effects on NSCLC xenografts by eradicating TICs. Hence, targeting α2δ1 to prevent calcium influx provides a novel strategy for targeted therapy against TICs of NSCLC.

Wnt/β-catenin Signaling Inhibitors suppress the Tumor-initiating properties of a CD44+CD133+ subpopulation of Caco-2 cells

Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44-CD133-, CD44-CD133+, and CD44+CD133+ subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44+CD133+ cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44+CD133+ Caco-2 cells contained mixed populations of CD44+CD133+ and non-CD44+CD133+ Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44+CD133+ subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44+CD133+ cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/β-catenin pathway was over-activated in CD44+CD133+ cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/β-catenin signaling inhibitors. Our findings suggest that the CD44+CD133+ subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development.

Cancer Stem Cell-Associated Pathways in the Metabolic Reprogramming of Breast Cancer.

Metabolic reprogramming of cancer is now considered a hallmark of many malignant tumors, including breast cancer, which remains the most commonly diagnosed cancer in women all over the world. One of the main challenges for the effective treatment of breast cancer emanates from the existence of a subpopulation of tumor-initiating cells, known as cancer stem cells (CSCs). Over the years, several pathways involved in the regulation of CSCs have been identified and characterized. Recent research has also shown that CSCs are capable of adopting a metabolic flexibility to survive under various stressors, contributing to chemo-resistance, metastasis, and disease relapse. This review summarizes the links between the metabolic adaptations of breast cancer cells and CSC-associated pathways. Identification of the drivers capable of the metabolic rewiring in breast cancer cells and CSCs and the signaling pathways contributing to metabolic flexibility may lead to the development of effective therapeutic strategies. This review also covers the role of these metabolic adaptation in conferring drug resistance and metastasis in breast CSCs.

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.

MTSS1 suppresses mammary tumor-initiating cells by enhancing RBCK1-mediated p65 ubiquitination.

Tumor-initiating cells (TICs) are considered the culprits of cancer development and progression. Dysregulation of metastasis suppressor protein 1 (MTSS1) has been widely observed in tumor metastasis, but its functional contribution and mechanism in cancer is poorly understood. Here we report a role of MTSS1 in suppressing TICs in breast cancer. Mtss1 knockout (KO) enhances the mammary epithelial TIC subpopulation in both luminal and basal-like breast cancer mouse models. MTSS1 also suppresses tumorsphere formation in breast cancer cells. Mechanistically, MTSS1 interacts with the E3 ligase RanBP2-type and C3HC4-type zinc finger containing 1 (RBCK1) to facilitate RBCK1-mediated p65 ubiquitination and degradation, thus suppressing the NF-κB signaling pathway and tumorigenesis. In addition, actin beta-like 2 (ACTBL2) competes with RBCK1 for MTSS1 binding, leading to p65 stabilization. Importantly, MTSS1 silencing promotes patient-derived organoid formation and xenograft growth. MTSS1 downregulation in clinical tumors is also linked to worse prognosis. Overall our data reveal a new paradigm of NF-κB regulation and may have important implications in therapeutics targeting TICs.

Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma

Hepatic resection and liver transplantation with adjuvant chemo- and radiotherapy are the mainstay of hepatocellular carcinoma (HCC) treatment, but the 5-year survival rate remains poor because of frequent recurrence and intrahepatic metastasis. Only sorafenib and lenvatinib are currently approved for the first-line treatment of advanced, unresected HCC, but they yield modest survival benefits. Thus, there is a need to identify new therapeutic targets to improve current HCC treatment modalities. The HCC tumor model was generated by hydrodynamic transfection of AKT1 and β-catenin (CTNNB1) oncogenes. Cancer cells with stemness properties were characterized following isolation using side population (SP) and CD44 surface markers by flow cytometry. The effect of Jak/Stat inhibitors was analyzed in vitro by using tumorsphere culture and in vivo using an allograft mouse model. Co-activation of both Wnt/β-catenin and Akt/mTOR pathways was found in 14.4% of our HCC patient cohort. More importantly, these patients showed poorer survival than those with either Wnt/β-catenin or Akt/mTOR pathway activation alone, demonstrating the clinical relevance of our study. In addition, we observed that Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through SP analysis and expression of the cancer stem cell-like marker CD44, which may contribute to tumor self-renewal and drug resistance. Consequently, we identified small molecule inhibitors of the Jak/Stat pathway that demonstrated efficacy in mitigating tumor proliferation and formation in Akt/β-catenin-driven HCC. In conclusion, we have shown that Akt/β-catenin tumors contain a subpopulation of tumor-initiating cells with stem/progenitor-like characteristics which can be effectively targeted with inhibitors of the Jak/Stat pathway, demonstrating that inhibition of the Jak/Stat pathway could be an alternative method to overcome drug resistance and effectively treat Akt/β-catenin-driven HCC tumors. The prognosis for patients with hepatocellular carcinoma is poor, partly because of the lack of effective treatment options for those with more advanced disease. In this study, we identified a subpopulation of cancer cells with stem cell-like properties that were critical for tumor maintenance and growth in a mouse model of hepatocellular carcinoma. Through further experiments, we demonstrated that the Jak/Stat pathway is a promising therapeutic target in hepatocellular carcinoma.

Transcriptomic analysis reveals that BMP4 sensitizes glioblastoma tumor-initiating cells to mechanical cues

The poor prognosis of glioblastoma (GBM) is associated with a highly invasive stem-like subpopulation of tumor-initiating cells (TICs), which drive recurrence and contribute to intra-tumoral heterogeneity through differentiation. These TICs are better able to escape extracellular matrix-imposed mechanical restrictions on invasion than their more differentiated progeny, and sensitization of TICs to extracellular matrix mechanics extends survival in preclinical models of GBM. However, little is known about the molecular basis of the relationship between TIC differentiation and mechanotransduction. Here we explore this relationship through a combination of transcriptomic analysis and studies with defined-stiffness matrices. We show that TIC differentiation induced by bone morphogenetic protein 4 (BMP4) suppresses expression of proteins relevant to extracellular matrix signaling and sensitizes TIC spreading to matrix stiffness. Moreover, our findings point towards a previously unappreciated connection between BMP4-induced differentiation, mechanotransduction, and metabolism. Notably, stiffness and differentiation modulate oxygen consumption, and inhibition of oxidative phosphorylation influences cell spreading in a stiffness- and differentiation-dependent manner. Our work integrates bioinformatic analysis with targeted molecular measurements and perturbations to yield new insight into how morphogen-induced differentiation influences how GBM TICs process mechanical inputs.

CD271 Confers an Invasive and Metastatic Phenotype of Head and Neck Squamous Cell Carcinoma through the Upregulation of Slug.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is comprised of heterogeneous populations of cells, and CD271 (NGFR; p75NTR) has been associated with a tumor-initiating cell subpopulation. This study assessed the role of CD271 in modulating metastatic behavior in HNSCC.Experimental Design: CD271 was overexpressed in murine and human oral squamous cell carcinoma cells to assess the impact of CD271 activation on the invasive and metastatic phenotype of these cells, using in vitro and orthotopic in vivo modeling. Treatment with human nerve growth factor (NGF) to activate CD271, as well as shRNA knockdown of the CD271-upregulated Snai2 expression, was used to assess the mechanism of the CD271-induced invasive phenotype. Relevance of CD271 expression in human HNSCC was evaluated in patient-derived xenografts (PDX) and primary human oral cancers, annotated with clinical behavior characteristics and survival data.Results: Forced expression of CD271 resulted in a more invasive and metastatic phenotype. Slug, an epithelial-to-mesenchymal transition (EMT)-related transcription factor, encoded by Snai2, was highly expressed in MOC2-CD271 and HSC3-CD271, compared with respective parental cells. CD271 activation by NGF conferred enhanced invasiveness in CD271-overexpressing cells, which was abrogated by Snai2 knockdown. In PDXs and primary human HNSCC, CD271 expression correlated with higher Snai2 expression, greater nodal metastasis, and shorter disease-free survival.Conclusions: Activation of CD271 results in upregulation of Snai2/Slug, which, in turn, results in a more invasive phenotype and an enhanced capacity for metastasis to regional lymph nodes. These findings point to CD271 as a promising, therapeutic target for oral cancer metastasis. Clin Cancer Res; 24(3); 674-83. ©2017 AACR.

Abstract 910: Genetic subclone heterogeneity of the human colon cancer initiating cell compartment

Abstract A subpopulation of tumor-initiating cells (TIC) drives colorectal cancer (CRC) progression in serial xenotransplantation. Strikingly, the CRC TIC compartment itself is heterogeneous and comprised of a hierarchy of long-term (LT-) TIC, tumor transient amplifying cells (T-TAC) and delayed contributing (DC-) TIC. Whether this functionally heterogeneous TIC compartment is genetically homogenous or whether distinct genetic subclones drive the functional heterogeneity of the TIC compartment is yet unknown. To address this question, we performed high coverage (91-126 fold) whole genome sequencing on primary CRC patient tumors (n = 3), corresponding serially passaged TIC enriched spheroids as well as spheroid derived serial xenografts. Sequenced samples harbored between 22.800 and 232.000 synonymous or non-synonymous single nucleotide variants (SNVs). In addition, all samples contained multiple focal or large-scale somatic copy number alterations (CNAs). Clustering of SNVs as well as subclonal copy numbers from serial xenografts and spheroids were used to define SNV- and CNA-based subpopulations. Next, cellular fractions of identified subpopulations were determined and combined applying maximal parsimony to create models of the minimal number of subclones present in each sample. Using this strategy, we found that multiple subclones were present in each sample analyzed. Subclone heterogeneity was maintained during serial in vitro passaging and serial xenografting. Importantly, tumor initiation in xenografts was driven by at least 3 distinct genetic subclones whose relative contribution dynamically changed over time. Strikingly, in serial samples from the same patients, different genetic subclones grew out in vivo and in vitro. To test whether functional heterogeneity of the TIC compartment is related to the presence of genetic subclones, we assessed the contribution of different TIC subtypes - LT-TIC, T-TAC and DC-TIC - at early and late time points of xenografting using secondary genetic marking. Therefore, 1×10⁁5 cells derived from early and late generation xenografts were transduced with an integrating lentiviral vector, thereby generating a stable barcode-like genetic mark which differs in each transduced cell. Following serial transplantation of transduced cells for 3 mouse generations, tumors were harvested and lentiviral integration sites were determined using highly sensitive LAM-PCR and high-throughput sequencing. Assessing the relative contribution of LT-TIC, T-TAC and DC-TIC revealed that the functional heterogeneity of the TIC compartment was preserved despite profound genetic subclone dynamics in serial xenotransplantation. These results strongly indicate that multiple genetic subclones drive long-term tumor formation and that functional heterogeneity of the CRC TIC compartment is not based on specific genetic subclones. Citation Format: Klara M. Giessler, Kortine Kleinheinz, Gnana Prakash Balasubramanian, Daniel Hübschmann, Taronish D. Dubash Rai, Sebastian D. Dieter, Christine Siegl, Christopher M. Hoffmann, Sarah Weber, Raffaele Fronza, Saira Afzal, Manfred Schmidt, Martin Schneider, Alexis Ulrich, Juergen Weitz, Wilko Weichert, Matthias Schlesner, Benedikt Brors, Claudia R. Ball, Hanno Glimm. Genetic subclone heterogeneity of the human colon cancer initiating cell compartment. [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 910.

Abstract 2769: Response to 5FU therapy in colorectal cancer is affected by EZH2-mediated BclX splicing

Abstract Background and Aims: The chromatin modifier EZH2 is remarkably up-regulated in many cancer types including colon cancer. High levels of EZH2 have been correlated with tumor metastasis and regulation of tumor-initiating cell subpopulation. Since, DZNep, an EZH2 inhibitor, can inhibit the growth capability of colon cancer by inducing apoptosis; down-regulation of EZH2 seems to be a promising therapeutic approach. Our preliminary data showed that EZH2 levels were remarkably increased in blood samples from metastatic colorectal cancer patients who did not respond to the combination therapy with bevacizumab-irinotecan-5FU in comparison to patients who were responders and developed a stable disease. Many apoptosis-related proteins possess a dual function (pro- and anti-apoptotic) due to alternative splicing. Therefore we aimed to find possible apoptosis-associated EZH2 targets to explain these interesting findings. Methods: HCT116 colon cancer cells were used for DZNep and 5FU treatment and its efficiency was verified by Western blot analysis. In vivo splicing assay was performed using BclX minigene construct. The mRNA expression of EZH2, caspase 2, Death-associated protein kinase (DAPK) and BclX isoforms was assessed by quantitative real-time PCR in cells (BclX also in blood of patients) and normalized to the GAPDH. Cell viability was examined by Crystal Violet staining. Results: Western Blot for caspase 3 cleavage indicated increased apoptotic levels after DZNep treatment. Combined DZNep and 5FU treatment of cells led to a synergistic effect in reducing cell viability. DZNep treatment specifically promoted pro-apoptotic BclXs isoform, but did not affect the shift of pro-apoptotic isoforms of caspase 2 and DAPK. Using a BclX minigene transfection we confirmed this effect exogenously. The ratio BclXs/Xl was significantly altered by overexpression of different splicing factors such as ASF/SF2, Sam68, after DZnep treatment. Moreover, patients who did not respond to the combination therapy had the highest EZH2 and BclXl isoform levels. Conclusion: Our data indicate a new interesting link between epigenetic regulation and the splicing machinery. Detailed investigation of EZH2-mediated regulation of BclXs/Xl ratio could have the potential for prediction of 5FU-based therapy response in colon cancer patients. Citation Format: Natalya Benderska, Maria Gazouli, Gerasimos Aravantinos, Arndt Hartmann, Regine Schneider-Stock. Response to 5FU therapy in colorectal cancer is affected by EZH2-mediated BclX splicing. [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 2769.

The Cancer Stem Cell Fraction in Hierarchically Organized Tumors Can Be Estimated Using Mathematical Modeling and Patient-Specific Treatment Trajectories.

Many tumors are hierarchically organized and driven by a subpopulation of tumor-initiating cells (TIC), or cancer stem cells. TICs are uniquely capable of recapitulating the tumor and are thought to be highly resistant to radio- and chemotherapy. Macroscopic patterns of tumor expansion before treatment and tumor regression during treatment are tied to the dynamics of TICs. Until now, the quantitative information about the fraction of TICs from macroscopic tumor burden trajectories could not be inferred. In this study, we generated a quantitative method based on a mathematical model that describes hierarchically organized tumor dynamics and patient-derived tumor burden information. The method identifies two characteristic equilibrium TIC regimes during expansion and regression. We show that tumor expansion and regression curves can be leveraged to infer estimates of the TIC fraction in individual patients at detection and after continued therapy. Furthermore, our method is parameter-free; it solely requires the knowledge of a patient's tumor burden over multiple time points to reveal microscopic properties of the malignancy. We demonstrate proof of concept in the case of chronic myeloid leukemia (CML), wherein our model recapitulated the clinical history of the disease in two independent patient cohorts. On the basis of patient-specific treatment responses in CML, we predict that after one year of targeted treatment, the fraction of TICs increases 100-fold and continues to increase up to 1,000-fold after 5 years of treatment. Our novel framework may significantly influence the implementation of personalized treatment strategies and has the potential for rapid translation into the clinic. Cancer Res; 76(7); 1705-13. ©2016 AACR.

Abstract A64: β-catenin as a TIC therapeutic target in epithelial ovarian cancer.

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women. The most common and lethal form of EOC which makes up greater than 75% of cases is high-grade serous cancer (HGSOC). Platinum-based drugs are used as first-line chemotherapeutics for all women diagnosed with HGSOC. Unfortunately, ~20% do not respond to this treatment and up to 80% of those patients which do respond recur within 5 years at which point the cancer is particularly difficult to eradicate. Emerging evidence supports the concept that platinum based therapies are able to eliminate the bulk of differentiated cancer cells but are unable to eliminate tumor-initiating cells (TICs) which are rare populations of tumor cells characterized by their ability to self-renew indefinitely and thus drive the expansion of drug-resistant tumors. Hence, there is immediate need of novel TIC targeted therapeutic approaches that can overcome chemo-resistance in EOC. Thus, identifying critical drivers of the stem-like phenotype that can be pharmacologically modulated in ovarian TICs is required. To date, no TIC targeting drugs have successfully completed clinical trials in EOC. Using primary HGSOC cells derived from HGSOC patient derived xenograft (PDX) models, we have recently identified β-catenin as a critical driver of HGSOC TIC phenotype in-vitro. β-catenin knockdown decreased stem-cell frequency and induced chemo-sensitivity in-vitro in platinum resistant HGSOC PDX derived primary cells. ALDH positive Ovarian TIC subpopulation with high Wnt reporter activity was platinum resistant and exhibited increased expression of β-catenin regulated stem-cell markers clearly identifying β-catenin regulated stem-cell transcriptional axis as a driver of chemo-resistance in Ovarian TICs. Accordingly, the β-catenin stem-cell transcriptional axis specific inhibitor iCG-001 (PRI-724) decreased stem-cell frequency and induced chemo-sensitivity in platinum resistant ovarian TICs, suggesting that pharmacologic inhibition of β-catenin could be a potential novel approach aimed at eradicating TICs in EOC. Furthermore, in-vivo tumor-initiation assays showed a robust decrease in tumor-initiation capacity upon β-catenin knockdown in platinum resistant HGSOC cells, thus establishing β-catenin as a driver of TIC phenotype in HGSOC. In sum, we have identified that pharmacologic inhibition of β-catenin can be a novel TIC targeted therapy approach to overcome chemo-resistance and improve patient outcome in in EOC. Our results suggest that the β-catenin specific inhibitor PRI-724, that has shown promising results in phase I clinical trials in solid tumors, can be a potential TIC targeting drug in EOC. These results will form a solid foundation for clinical trials with PRI-724 in patients with chemo-recurrent EOC. Citation Format: Anil Belur Nagaraj, Peronne Joseph, Olga Kovalenko, Sareena Singh, Amy Armstrong, Analisa DiFeo. β-catenin as a TIC therapeutic target in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A64.

CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma.

Tumor-initiating cells (TICs) in squamous cell carcinoma of the head and neck (SCCHN) are best characterized by their surface expression of CD44. Although there is great interest in identifying strategies to target this population, no marker of these cells has been found to be functionally active. Here, we examined the expression of the purported marker of normal human oral epithelial stem cells, CD271. We show that CD271 expression is restricted to a subset of the CD44+ cells. Using xenograft assays, we show that the CD44+CD271+ subpopulation contains the most tumorigenic cells. Loss of CD271 function results in a block in the G2-M phase of the cell cycle and a profound negative impact on the capacity of these cells to initiate tumor formation in vivo. Incubation with recombinant NGF results in enhanced phosphorylation of Erk, providing additional evidence that CD271 is functionally active. Finally, incubation of SCCHN cells with antibody to CD271 results in decreased Erk phosphorylation and decreased tumor formation in vivo. Thus, our data are the first to demonstrate that CD271 more specifically identifies the TIC subpopulation within the CD44+ compartment in SCCHN and that this receptor is a functionally active and targetable molecule.

PIAS1 Regulates Breast Tumorigenesis through Selective Epigenetic Gene Silencing

Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.

Identification of a Novel Subpopulation of Tumor-Initiating Cells from Gemcitabine-Resistant Pancreatic Ductal Adenocarcinoma Patients

Pancreatic ductal adenocarcinoma is highly resistant to systemic chemotherapy. Although there are many reports using pancreatic cancer cells derived from patients who did not receive chemotherapy, characteristics of pancreatic cancer cells from chemotherapy-resistant patients remain unclear. In this study, we set out to establish a cancer cell line in disseminated cancer cells derived from gemcitabine-resistant pancreatic ductal adenocarcinoma patients. By use of in vitro co-culture system with stromal cells, we established a novel pancreatic tumor-initiating cell line. The cell line required its direct interaction with stromal cells for its in vitro clonogenic growth and passaging. Their direct interaction induced basal lamina-like extracellular matrix formation that maintained colony formation. The cell line expressed CD133 protein, which expression level changed autonomously and by culture conditions. These results demonstrated that there were novel pancreatic tumor-initiating cells that required direct interactions with stromal cells for their in vitro cultivation in gemcitabine-resistant pancreatic ductal adenocarcinoma. This cell line would help to develop novel therapies that enhance effects of gemcitabine or novel anti-cancer drugs.

Abstract IA09: Targeting tumor-initiating cells in xenograft models of human breast cancer

Abstract Historically, investigation of treatment resistance in breast cancer has focused on the identification of acquired genetic or epigenetic alterations in tumor epithelial cells that confer resistance to specific agents, or to multiple agents. Our recent data indicate that a unique subpopulation of tumor-initiating cells (TIC), or “cancer stem cells” present in the original tumors appear to possess a form of intrinsic (de novo) resistance to conventional systemic therapies including radiation and chemotherapies, and are therefore likely responsible for tumor initiation and cancer re-growth. Agents that may target TIC cells specifically are becoming attractive experimental therapeutic candidates. Current markers that allow enrichment of breast cancer TIC by fluorescence activated cell sorting (FACS) (e.g. CD44+/CD24low/neg and ALDH activity) are not universally expressed, cannot be monitored in vivo, and typically identify a much larger percentage of cells than the percentage of cells estimated to serve as TIC. Thus, to evaluate TIC therapeutic targeting effectively, new markers must be identified. To investigate the location and function of TIC, and to evaluate agents that may specifically target them therapeutically, we are developing a series of lentiviral fluorescent signaling reporters to identify cells responsive to Wnt, Hedgehog, Notch, and Stat3-mediated signaling – four pathways implicated in TIC self-renewal or regulation. These reporters have been introduced into a recently developed bank of patient-derived human breast cancer xenograft (PDX) models established in our laboratory, as well as traditional cell line xenograft models. Results thus far indicate that both Wnt responsiveness and STAT3-mediated signaling allows significant enrichment for the TIC population by FACS, and has allowed localization of responsive cells in vivo. Given that these pathways have been implicated in TIC regulation, we have begun to target these pathways using a battery of experimental therapeutics both in the preclinical setting, and in the case of Notch signaling, also in the clinical setting. Current data suggest that inhibition of Notch, STAT3, and Hedgehog signaling can enhance the efficacy of systemic chemotherapy, and can impair or alter the function of TIC in limiting dilution transplantation assays. However, in the case of Hedgehog signaling inhibition, the transplantation behavior of the TIC fraction is biphasic suggesting a novel form of inhibitory cell-cell interaction is present in at least some tumors. Overall, results are consistent with the concept that TIC targeted therapeutics may ultimately prove useful clinically. However, our results also indicate that identification and use of additional agents that actually kill TIC will likely be required for eradication of TIC in patients. Citation Format: Michael T. Lewis. Targeting tumor-initiating cells in xenograft models of human breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA09.

Retraction Note: Marker-independent identification of glioma-initiating cells

In the version of this article initially published, the authors described a method to identify tumor-initiating cells from human primary gliomasphere cell cultures or directly from fresh human glioma specimens. They used FACS analysis to describe a tumor-initiating cell subpopulation that displayed a specific morphology (high forward scatter, low side scatter) and had high fluorescence in the FL1 channel of the FACS (excitation at 488 nm, emission at 520 nm).