The second International Cancer Stem Cell Conference in Cleveland, Ohio, on September 20–23, 2016, convened 330 attendees from academic, industrial, and clinical organizations. It featured a debate on the concepts and challenges of the cancer stem cells (CSC) as well as CSC-centered scientific sessions on clinical trials, genetics and epigenetics, tumor microenvironment, immune suppression, metastasis, therapeutic resistance, and emerging novel concepts. The conference hosted 35 renowned speakers, 100 posters, 20 short talks, and a preconference workshop. The reported advances of CSC research and therapies fostered new collaborations across national and international borders, and inspired the next generation's young scientists. Cancer Res; 77(19); 5222–7. ©2017 AACR.
Cancer Stem Cell Overview
Over the last 20 plus years, cancer stem cells (CSC) have been functionally identified in human leukemia (1) and many solid tumors, such as breast, ovarian, prostate, brain, colon, lung, and others. Increasing evidence supports that CSCs remain the root of cancer, seeds of metastasis, and sources of therapy resistance (2). Although the concept of CSCs has provided an opportunity to assess the complexity of cancer using a developmental-biology–inspired paradigm, the big question remains to what level and how CSCs would affect cancer medicine. CSC Conference 2016 provided a forum to challenge and foster the forefront research and clinical applications of CSCs.
At the opening session, a forum debate about the beliefs and challenges on CSCs followed between two groups, including the believers Drs. John E. Dick, Luis Parada, and Tannishtha Reya, and the challengers Drs. Mina Bissell, Geoffrey M. Wahl, and Yogen Saunthararajah. The heated debate covered the definition, impact, and clinical implications of CSCs in cancer medicine, and more. On an achieved consensus, CSCs are functionally identified by their self-renewal and tumorigenic capacity, whereas heterogeneous markers may be used to enrich CSCs across cancers. Both intrinsic and extrinsic signaling pathways from genetic, epigenetic, and microenvironmental alterations converge to regulate stemness of cells, thereby featuring the plasticity of CSCs. Stemness signature genes are clearly associated with clinical outcomes of cancer patients (3), but strategies targeting CSCs would need to be combined with other targeted and immunotherapies to eradicate cancer and achieve durable disease status.
The keynote speakers set up the high standard of the state-of-the art research reports as well as challenges to the CSC field. Dr. John E. Dick (University Health Network, Toronto, Ontario, Canada), who pioneered the CSC field by first identifying human leukemia stem cells (LSC; ref. 1) and colon CSCs, shared the dark side of stem cells (SC) where his latest research has identified that a preleukemic SC with DNMT3A mutations may be the first step in initiating disease and also the culprit evading therapy and triggering relapse in patients with AML (4). Dr. Robert Weinberg (Massachusetts Institute of Technology, Cambridge, MA) described normal and neoplastic SCs and the epithelial–mesenchymal transition (EMT) program. EMT transcription factors, such as Slug, Snail, Sox9, and Zeb1, cooperatively act to determine the mammary SC state and CSC plasticity (5). Dr. Luis Parada (Memorial Sloan Kettering, New York, NY) emphasized that CSCs are defined by function not by epitopes or surrogate assays. His work explored the stem cell origin of CSCs in malignant glioma and utilized the CDG transgene in specific promoter elements to target both CSCs and transit-amplifying cells. This discovery implicates that same genetic drivers in different cells of origin develop distinct glioblastoma multiforme (GBM) tumor types (6).
Clinical Trials of CSC Targeting Therapeutics
The evolution of clinical trials that target CSCs holds promise of affecting cancer medicine. CSC functions have been linked to dysregulated stem cell pathways such as Wnt, Notch, and Hedgehog signaling (9), which are fundamental for normal SCs. Despite the great challenges to specifically target CSCs, two hedgehog pathways inhibitors targeting SMO (LDE225/sonidegib and GDC-0449/vismodegib) have received FDA approval for treating basal cell carcinoma (10). Notable approaches have been developed to target cancer-specific fusion receptors (11) and CD47 (12). Ongoing CSC-targeting clinical trials are being conducted to evaluate their efficacy in a variety of cancers.
Dr. Max S. Wicha (University of Michigan, Ann Arbor, MI) discussed the therapeutic targeting of breast CSCs and outlined some of the therapeutic agents currently used in clinical trials including demcizumab (anti-Notch ligand DLL4 antibody), ipafricept (Fzd8 fusion protein OMP-54F28), vantictumab (anti-Frizzled), reparixin (CXCR1 inhibitor), defactinib [focal adhesion kinase (FAK) inhibitor], tarextumab (OMP-59R5), and BBI608 (targets STAT3). His small-molecule and high-throughput siRNA screenings also discovered novel agents that target CSC regulatory pathways (13). Dr. Jonathan Pachter (Verastem, Inc.) presented CSC-targeting strategies in clinical trials using selective inhibitors of FAK and PI3K/mTOR (14). Although targeting CSC alone may not be sufficient to remove the bulk tumor, combining FAK inhibitors with other therapies such as the immune checkpoint blocking antibodies is a promising strategy that is currently being tested in several clinical trials (ClinicalTrials.gov NCT02546531, NCT02758587, NCT02943317). CSC-targeting ChemoID drug response assays served as correlative endpoints and stratification variables for glioblastoma (Pier Paolo Claudio, University of Mississippi, Oxford, MS; ref. 15), which could lead to more efficient and personalized anticancer therapy in the future. FABP5 drives self-renewal of triple-negative breast CSCs by enhancing the transcriptional activity of PPARδ to induce NANOG, SOX2, and OCT4 (Dr. Liraz Levi, Cleveland Clinic Foundation, Cleveland, OH). FABP5 inhibitor-F19 was developed to decrease tumorigenesis and stemness (16).
Although different therapeutic strategies targeting CSCs are being evaluated in clinical trials, combination therapies consisting of existing agents and CSC pathway inhibitors are to be optimized to affect cancer care. Furthermore, high-throughput screenings may help better determine the best personalized approach to treat patients. In addition, examining genomic alterations at premalignant stages (6, 7) could identity malignancy-specific targets.
Genetics, epigenetics, and development
Accumulation of genetic and epigenetic alterations contributes to the self-renewal and drug-resistant capacity of CSCs. Dr. Connie Eaves (BC Cancer Agency), a keynote speaker, reported the clonal analysis of human breast cancer origins and progression, using cellular DNA barcoding with multiplexed, high-resolution techniques to better study the heterogeneity of human malignant tumors (17, 18). The frequencies of clone-initiating cells vary from 1/10 to 1/10,000, and the carcinogenic process in human normal epithelia requires the acquisition of multiple driver mutations (18).
As discussed in the conference, cancer arises from the populations with self-renewal and multipotent properties. Dr. Geoffrey M. Wahl (Salk Institute, La Jolla, CA) examined the plasticity of the fetal mammary SCs (fMaSC) as their transcriptome is significantly enriched in basal-A human breast cancers. They exhibit characteristics of both luminal and basal cells. While their transcriptomes are different from ‘CSC,’ they can be induced to undergo an atypical EMT that is also reversible and reflects that found in metastatic breast cancers. Differentiation by inflammation, oncogenes, or both, likely will present challenges to therapies intended to selectively target CSCs (19). In addition, Dr. Benjamin Spike (University of Utah, Salt Lake City, UT) discussed the SC transcriptional continuum in mammary development and implications for CSCs with embryonic SC and fMaSC-like signatures. His studies also showed that MCAM, Cripto-GRP78 pathways regulate breast cancer development (20).
Moreover, Dr. Xiling Shen (Duke University, Durham, NC) addressed how asymmetric division determines cell fate, SC or non-SC, through miR-34a-Numb-Notch signaling cascade. A long noncoding RNA suppresses miR-34a by recruiting DNA methyltransferase Dnmt3a via prohibitin-2 and histone deacetylase 1 (161). Dr. Marcus E. Peter (Northwestern University, Chicago, IL) reported that chronic stimulation of CD95 results in lower levels of miR-200c, monitored using a sensor plasmid for miR-200c, increasing the CD24 low stem-like population in breast cancer cells (22). This is driven by a type I interferon/STAT1 pathway (23). Dr. Dean Tang (Roswell Park Cancer Institute, Buffalo, NY) emphasized NANOG-mediated reprograming in prostate cancer (24). Dr. Marius Wernig (Stanford University, Stanford, CA) demonstrated that ASCL1, MYT1L, and BRN2 mediate direct conversion reprogramming of mouse embryonic fibroblasts and other cell types to functional neurons bypassing the induced pluripotent stem cells. His group found that ASCL1 is sufficient to generate functional neurons, whereas MYT1l (repressing nonneuronal programs) and BRN2 enhance the maturation process (25).
A few short talks updated that DDB2 can limit the ovarian CSC population, probably through suppressing the transcription of ALDH1A1 as a transcription regulator (Qi-En Wang, Ohio State University, Columbus, OH; ref. 26). OLIG2+ marks SOX2+ quiescent SCs and mediates medulloblastoma growth through altering the epigenetic landscape to activate oncogenic pathways in adult mice (Q. Richard Lu, Cincinnati Children's Hospital, Cincinnati, OH). PRMT6- dependent CRAF/ERK signaling regulates CSC plasticity in liver cancer (Stephanie Ma, University of Hong Kong, Hong Kong). A subset of connexin proteins drives self-renewal in triple-negative breast cancer by forming an aberrant intracellular complex with FAK and NANOG (Praveena Thiagarajan, Cleveland Clinic Foundation).
Although both intrinsic and extrinsic factors merge to frame CSC plasticity, microenvironment influences play a pivotal role in the signaling cascades during CSC progression and differentiation. Based on the pioneering work of keynote speaker Dr. Mina Bissell (Lawrence Berkeley National Laboratory, Berkeley, CA), tumor growth and malignant behavior are regulated at the level of tissue organization and dependent on the extracellular matrix. Extracellular glucose determines malignant tumor phenotype via EPAC/RAP1 and O-GlcNAc pathways (27). The p53 association with laminin 5 induces dormancy of cancer cells, resulting in drug resistance. In GBM CSC studies, Dr. Jeremy Rich (Cleveland Clinic Foundation) concluded that various microenvironmental niche factors, such as acidic conditions, glucose restriction, and iron metabolism, modulate CSC functions. CSC-targeting strategies would be specifically tailored to the microenvironment.
Keynote speaker Dr. Carla F. Kim (Boston Children's Hospital, Boston, MA) spearheaded successful three-dimensional (3D) cocultures of bronchioalveolar SCs (BASC) with endothelial cells and demonstrated that endothelium-derived Tsp1 influences BASC alveolar differentiation under control of Bmp4-stimulated NFATc1 in a lung endothelium-specific manner (28). In addition, her recent work revealed the chromatin regulation of lung adenocarcinoma tumor-propagating cells and BASCs via H3K9 methyltransferases G9a/Glp. Dr. Antonio Iavarone (Columbia University, New York, NY) described a mechanism where hypoxia drives the CSC state in GBM through inhibitor of differentiation (ID), which is often amplified in both adult and pediatric gliomas for tumor maintenance or upregulated in hypoxic conditions through the PHD1–DYRK1–ID2 signaling axis (29). His group developed a peptide that blocks the interaction between ID2 and the VHL ubiquitin ligase complex.
Additional short talks further emphasized the role of microenvironmental signals in CSC regulation. Oncostatin-M in normal human epithelial cells induces senescence marker GLB1 and Snail expressions, which are related to EMT and CSC properties (Benjamin Bryson, Case Western Reserve University, Cleveland, OH). Additional mechanisms include oncostatin-M–driven, Zeb1-dependent induction of EMT and CSC properties in pancreatic ductal adenocarcinoma (Neetha Parameswaran, Case Western Reserve University), cancer-associated fibroblast regulation on tumor-initiating cell (TIC) plasticity in hepatocellular carcinoma (Yuen-Ting Eunice Lau, University of Hong Kong; ref. 30), and stearoyl-CoA desaturase as a mediator of TICs in hepatospheres (Terence Kin-Wah Lee, The Hong Kong Polytechnic University, Hong Kong). Based on a mouse model for non–small cell lung carcinoma, the alterations in the KEAP1/NRF2 pathway may serve as predictive biomarkers for personalized therapies against non–small cell lung cancers (Youngtae Jeong, Stanford University; ref. 31).
Immune suppression and stemness
Studying the innate and adaptive immune response to cancer cells and CSCs has promoted one of the most promising therapeutic approaches in the cancer field—immunotherapy. Dr. Thomas F. Gajewski (University of Chicago, Chicago, IL) introduced T-cell inflamed versus noninflamed tumor microenvironments in melanoma. A CD8+ T-cell infiltrate is ineffective at eradicating tumor cells unless the immune-suppressive Tregs, PD1/PD-L1, and IDO (tryptophan metabolizing enzyme) are inhibited in combination. His work demonstrated that melanoma-intrinsic β-catenin activation or PTEN loss prevents host antitumor immune response with reduced T-cell dynamics (recruitment), a lack of T-cell–inflamed phenotype, and resistance to anti–PD-1 immunotherapy (32). Dr. Dean W. Felsher (Stanford University) highlighted that the MYC oncogene regulates immune checkpoints through inducing CD47 and PD-L1 expression (33). MYC inactivation enhanced antitumor immune response; therefore, reducing MYC expression and activity may restore immune response against cancer cells (33).
Dr. Shideng Bao (Cleveland Clinic Foundation) described the interplay between glioma SCs (GSC) and tumor-associated macrophages (TAM). GSCs recruit tumor-supportive TAMs (M2) by secreting periostin, which binds to αvβ3 integrin on TAMs (34). Dr. Jeffrey Rosen (Baylor College of Medicine, Houston, TX) emphasized the cooperative role of WNT and FGF signaling pathways in the immune microenvironment in tumor dormancy, recurrence, and metastasis. Inhibition of FGF receptor leads to regression of Wnt1/iR1 tumors and decreases recruitment of myeloid-derived suppressor cells (MDSC), promoting EMT, tumor invasion, angiogenesis, and metastasis.
Dr. Justin D. Lathia (Cleveland Clinic Foundation) identified CSC-driven and immune-suppressive MDSCs in the brain of GBM patients. CSC-derived MIF in GBM suppresses immune rejection by recruiting MDSCs via immune suppressive enzyme arginase-1 in CXCR2-dependent manner (35). In addition, TLR4 decreases SC properties in CSCs by controlling retinoblastoma-binding protein 5 in GBM CSCs. In a separate study, autologous dendritic cells loaded with antigens from autologous ovarian CSCs serve as an adjunctive treatment for advanced ovarian cancer (Bob Dillman, AVITA Biomedical).
Therapeutic resistance and heterogeneity
The role of CSCs in therapy resistance has been well established based on previous studies (36). Dr. Tannishtha Reya (University of California San Diego, San Diego, CA) presented her work on SC signals in cancer heterogeneity and therapy resistance. She reported that the SC determinant Musashi is a critical driver of pancreatic cancer progression, and Musashi reporters are a distinctive tool to identify therapy resistance (37). Dr. Peter Dirks (Hospital for Sick Children, Toronto, Ontario, Canada) reported that driving GBM cells into terminally differentiated neuronal lineage is important to reduce resistance. Dr. Keith Chan (Baylor College of Medicine) reported that COX2-mediated PGE2 release contributes to recruitment of CK14+ cells following treatment, and that blockade of PGE2 release inhibits the accumulation of resistance to bladder cancer therapy (38). In addition, CD117 is a sensitive marker for tumor grade and recurrence in prostate cancer (Bethany Kerr, Wake Forest University; ref. 39). Inhibition of CD55 holds great promise as a CSC-targeted therapy for cisplatin-resistant endometrioid tumor patients (Caner Saygin, Cleveland Clinic Foundation). Novel connexin-43 mimetic peptide (JM2) decreases connexin-43–mediated microtubule interaction in GSCs and reduces temozolomide resistance and neurosphere formation (Samy Lamouille, Virginia Tech Carillion Research Institute, Roanoke, VA; ref. 40). A 3D GBM-CSC organoid culture system has been developed to preserve the cell heterogeneity and microenvironmental gradients in patient tumors and will aid in the development of new therapies to treat the heterogeneity of GBM (Chris Hubert, Cleveland Clinic Foundation; ref. 41).
Metastasis, novel technologies, and new concepts
Increasing evidence supports the mediation of CSCs in metastasis. The detection of a subpopulation of circulating tumor cell (CTC) with putative stem cell progenitor markers in patients with metastatic breast cancer and liver cancer implicates circulating CSCs in metastasis. Lgr5+ colorectal CSCs were found to be mediating cancer metastasis (42). Increased metastatic potential was later observed in breast cancer patient–derived CTCs expressing putative CSC markers (43). Dr. Arnold I. Caplan (Case Western Reserve University) emphasized the molecular control of melanoma metastasis into bone, demonstrating that mesenchymal stem cell/pericyte presence is required for efficient melanoma cancer cell intravasation and extravasation to bone (44). A notable study was shared by Dr. Simona Parrinello (Imperial London College, London, United Kingdom) that in a murine GBM model, ephrin-B2 drives perivascular invasion and proliferation of glioma stem-like cells (45).
Recent evidence suggests that CTC clusters are present in the blood of cancer patients and correlated with metastasis and drug resistance in various cancers (46). CTC clusters showed 23- to 50-fold increase in metastatic ability compared with single CTCs (46). Dr. Huiping Liu (Case Western Reserve University) studies cluster formation of CTCs in breast cancer and discussed the role of breast CSCs in metastasis using PDX models. Her team demonstrated the cellular and molecular mechanisms (such as miR-206) via which CSCs promote metastasis (36, 47). Dr. Sumanta Goswami (Yeshiva University, New York, NY) showed that Wnt mediates a cross-talk between tumor microenvironment of metastasis and CSCs, suggesting a potential role of CSCs in metastasis.
A few presentations featured recent advances in new technologies to capture CTCs and image CSCs as well as in understanding of CSC-mediated recurrence and therapy resistance. CTC detection in the clinical samples has been challenging. Dr. Sunitha Nagrath's lab (University of Michigan) developed the GO-Chip made of graphene oxide and antibody-coated microposts to rapidly separate CTCs from blood samples (48). She demonstrated that 50% of patients with lung cancer were found to have CTCs in clusters, and their initial data show that these clusters correlate with survival. A new minimally invasive technique of intravital imaging using a lung window visualized the fate and microenvironment of tumor cells and CSCs in lung metastasis (Sonia Voiculescu, Albert Einstein College of Medicine, New York, NY). An optimized CTC isolation technique was employed to establish three cell lines of circulating colorectal cancer cells with CSC hallmarks and chemoresistance (Julie Pannequin, Institute of Functional Genomic, Montpellier, France). A newly designed gold nanoparticle, Smart Flare, was coated with a probe to bind mRNA transcripts of interest in the living cells as well as in CSCs (Steven McClellan, University of South Alabama, Mobile, AL).
Dr. Stanton L. Gerson (Case Western Reserve University and University Hospitals) reported that autologous transplants of hematopoietic progenitor cells with mutant O6-alkylguanine DNA alkyltransferase (MGMT) (P140K) overcome the side effects of combination therapies for GBM with temozolomide and MGMT inhibitor O6-benzylguanine (BG). This approach will allow patients to tolerate temozolomide and BG treatment with minimal bone marrow toxicity due to the coupled low affinity of P140K mutant to BG. Preliminary results demonstrated that this chemoprotection strategy is tolerable and safe.
Recognition of young scientists and career development workshop
The young investigator awards recognized five outstanding scientists. Their presentations featured (i) molecular mechanisms of CTC clustering in breast cancer (Xia Liu, Case Western Reserve University); (ii) a 3D culture system to model ovarian tumor therapeutic resistance (Geeta Mehta, University of Michigan); (iii) CSC regulation of MDSC functions in GBM immune evasion (Daniel Silver, Cleveland Clinic Foundation; ref. 35); (iv) PKA regulation of tumorigenicity and chemotherapy resistance through activating PHF2 in immortalized human mammary cells (Diwakar Pattabiraman, Massachusetts Institute of Technology; ref. 49); and (v) genetic imprinting of CpG methylation state influences Hippo signaling through miR-127-SETD8-LAT2 pathway in stem/progenitor cells (Maider Zabala Ugalde, Stanford University).
At the beginning of the preconference workshop, an NIH grant workshop with talks from Dr. Michael Espey (NIH, Bethesda, MD), Dr. Nywana Sizemore (NIH), and Dr. Ming Lei (NIH) covered trainee career development strategies, discussion of grant opportunities, and career options discussions. At the Meet-the Editors session, Dr. Christine Weber and Dr. Deborah J. Sweet gave a brief overview of publishing in the Nature journals and Cell Press, respectively.
Although the link between developmental and cancer biology has long been appreciated, the assessment of stem cell programs in cancer has provided insight into cancer progression and provided opportunities to develop next-generation cancer therapies. Cellular heterogeneity and plasticity represent significant challenges in understanding stem cell biology. Given the intrinsic attributes of CSCs and stemness signaling shown to suppress immune response and mediate immunotherapy resistance, we expect that targeting CSCs and modulating the tumor microenvironment to augment immune attacks on cancer would be a promising strategy to combine with existing approaches against cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We acknowledge the National Center for Regenerative Medicine and Case Comprehensive Cancer Center for hosting the conference. We appreciate the executive administrative team led by Charlene Mitchell who organized and made the conference a successful platform for scientific exchange and new initiations of breakthrough research and clinical applications.
The CSC 2016 and the meeting report were partially supported by NIH/NCIR13CA206377 (S.L. Gerson), P30 CA043703 (S.L. Gerson), and R00CA160638 (H. Liu).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.