Non-stem Cell Populations Research Articles

Doxorubicin-induced senescence promotes stemness and tumorigenicity in EpCAM-/CD133- nonstem cell population in hepatocellular carcinoma cell line, HuH-7.

The therapeutic induction of senescence is a potential means to treat cancer, primarily acting through the induction of a persistent growth‐arrested state in tumors. However, recent studies have indicated that therapy‐induced senescence (TIS) in tumor cells allows for the prolonged survival of a subgroup of cells in a dormant state, with the potential to re‐enter the cell cycle along with an increased stemness gene expression. Residual cells after TIS with increased cancer stem cell phenotype may have profound implications for tumor aggressiveness and disease recurrence. Herein, we investigated senescence‐associated stemness in EpCAM+/CD133+ liver cancer stem cell and EpCAM−/CD133− nonstem cell populations in HuH7 cell line. We demonstrated that treatment with doxorubicin induces senescence in both cell populations, accompanied by a significant increase in the expression of reprogramming genes SOX2, KLF4, and c‐MYC as well as liver stemness‐related genes EpCAM, CK19, and ANXA3 and the multidrug resistance‐related gene ABCG2. Moreover, doxorubicin treatment significantly increased EpCAM + population in nonstem cells indicating senescence‐associated reprogramming of nonstem cell population. Also, Wnt/β‐catenin target genes were increased in these cells, while inhibition of this signaling pathway decreased stem cell gene expression. Importantly, Dox‐treated EpCAM−/CD133− nonstem cells had increased in vivo tumor‐forming ability. In addition, when SASP‐CM from Dox‐treated cells were applied onto hİPSC‐derived hepatocytes, senescence was induced in hepatocytes along with an increased expression of TGF‐β, KLF4, and AXIN2. Importantly, SASP‐CM was not able to induce senescence in Hep3B‐TR cells, a derivative line rendered resistant to TGF‐β signaling. Furthermore, ELISA experiments revealed that the SASP‐CM of Dox‐treated cells contain inflammatory cytokines IL8 and IP10. In summary, our findings further emphasize the importance of carefully dissecting the beneficial and detrimental aspects of prosenescence therapy in HCC and support the potential use of senolytic drugs in HCC treatment in order to eliminate adverse effects of TIS.

Radiation induces an inflammatory response that results in STAT3-dependent changes in cellular plasticity and radioresistance of breast cancer stem-like cells

Purpose: Pro-inflammatory cytokines within the tumor microenvironment, such as IL-6, contribute to the maintenance of stem cells and promote their survival following treatment. The IL-6/STAT3 pathway is a key regulator of genes involved in cancer progression. Activation of STAT3 promotes expansion of cancer stem cells in triple negative breast cancer. Radiation has also been shown to expand cancer stem cell populations and can induce stemness in nonstem cells. However, the role of IL-6/STAT3 in radiation-induced changes in cellular plasticity is unclear.Materials and methods: Expression and secretion of IL-6 from triple-negative breast cancer cell lines SUM159PT and MDA-MB-231 were determined after radiation treatment by real-time PCR and ELISA. Activation of STAT3 after radiation was determined by western blotting. Changes in cellular plasticity induced by radiation were determined by examining ALDEFLUOR activity, gene expression analysis of aldehyde dehydrogenase isoforms and mammosphere forming assays with and without the addition of STAT3 inhibitors. To determine the effect of radiation on nonstem cell populations, experiments were also carried out in ALDEFLUOR sorted cells.Results: Radiation induced an inflammatory response in both cell lines that resulted in activation of STAT3. Additionally, radiation induced a stem-like state as evidenced by an increased activity and expression of the ALDH isoforms ALDH1A1 and ALDH1A3, and increased self-renewal capabilities. Radiation increased ALDH activity and self-renewal in non-stem cell (ALDH−) populations, suggesting radiation-induced cellular reprograming. However, inhibition of STAT3 blocked the radiation-induced stem-like state in both ALDEFLUOR positive and negative populations, and enhanced radiosensitivity.Conclusions: Radiation-induced changes in cellular plasticity are STAT3 dependent and may be a potential target to reduce radioresistance in TNBC and improve treatment outcome.

STEM-26. ALTERED LIPID METABOLISM MARKS GLIOBLASTOMA STEM AND NON-STEM CELLS IN SEPARATE TUMOR NICHES

Abstract Clinical glioblastoma is marked by a strikingly heterogeneous mix of cell types, cellular metabolisms, and cellular microenvironments spread in different spatial locations throughout a tumor. We have created 3-dimensional organoid models that partially mimic the transition zone between nutrient-rich cellular tumor regions and nutrient-poor psuedopallisading and perinecrotic tumor zones. We found a dramatic disparity in lipid droplet presence between these regions with high lipid accumulation in the hypoxic organoid cores of a wide spectrum of patient derived specimens. This is accompanied by regionally restricted upregulation of HILPDA gene expression in the cores of our models, in clinical GBM specimens but not lower grade brain tumors, and localized specifically to pseudopallisading regions of patient tumors. We further show that lipid droplet accumulation overall marks perinecrotic and pseudopallisading regions in clinical GBM, indicating broadly altered lipid metabolism between these distinct cell populations. High lipid droplet accumulation is largely restricted to the non-stem cell populations of GBM organoids and sorted xenograft tumors whereas the stem cells are lipid-poor, suggesting lipid levels may not be simply a product of the microenvironment but also may be a reflection of cell state. We performed global lipidomic analysis on prospectively sorted stem and non-stem cells of multiple patient-derived models and found that GBM stem cells have comparatively decreased levels of neutral lipids, indicating a significant metabolic shift compared to non-stem cells from the same patient. Conversely, GBM stem cells have significantly increased levels of rare specific lipid species, and also display altered phospholipid synthesis and species specific alterations in phospholipid classes. Our findings suggest avenues for therapy by targeting the altered lipid metabolic pathways of these disparate tumor cell populations.

Modeling tumorspheres reveals cancer stem cell niche building and plasticity

Cancer stem cells have been shown to be critical to the development of a variety of solid cancers. The precise interplay mechanisms between cancer stem cells and the rest of a tissue are still not elucidated. To shed light on the interactions between stem and non-stem cancer cell populations we develop a two-population mathematical model, which is suitable to describe tumorsphere growth. Both interspecific and intraspecific interactions, mediated by the microenvironment, are included. We show that there is a tipping point, characterized by a transcritical bifurcation, where a purely non-stem cell attractor is replaced by a new attractor that contains both stem and differentiated cancer cells. The model is then applied to describe the outcome of a recent experiment. This description reveals that, while the intraspecific interactions are inhibitory, the interspecific interactions stimulate growth. This can be understood in terms of stem cells needing differentiated cells to reinforce their niches, and phenotypic plasticity favoring the de-differentiation of differentiated cells into cancer stem cells. We posit that this is a consequence of the deregulation of the quorum sensing that maintains homeostasis in healthy tissues.

Abstract 4837: Role of Notch signaling in human mucoepidermoid carcinoma and combined targeting of Notch and EGFR signaling as an anti-cancer strategy

Abstract Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy and currently no targeted therapy is available for MEC patients. MEC is characterized by a unique chromosomal translocation t(11;19)(q14-21;p12-13) that generates the CRTC1-MAML2 fusion. Our understanding of CRTC1-MAML2-mediated tumorigenesis and maintenance remains limited. MEC is considered a disease of stem/progenitor cells; however, the regulation of human MEC stem/progenitor cells has not yet been characterized. In this study, we focused on Notch signaling, a pathway important in regulating normal and cancerous stem cells, and investigated its regulatory role in human MEC cancer stem/progenitor cells as well as its potential therapeutic potential. First, we determined the status of Notch signaling activation in MEC cells. Western blotting showed the presence of cleaved active NOTCH1 and the expression of Notch downstream target gene HES1 in human CRTC1-MAML2 fusion-positive MEC cells, indicative of active Notch signaling in MEC. Second, we utilized two approaches of blocking endogenous Notch signaling in human MEC cells in vitro and in vivo, i.e. dnMAML1, a pan-Notch inhibitor that blocks the formation of the Notch transcriptional activation complex, and dibenzazepine (DBZ), a γ-secretase inhibitor that interferes with Notch receptor processing. We observed that Notch signaling inhibition had no effect on the proliferation of bulk MEC cells, but significantly reduced oncosphere forming capacity and ALDH-positive population in human MEC in vitro, suggesting that Notch signaling contributes to the maintenance of MEC stem/progenitor cells. Further, Notch signaling inhibition significantly reduced the growth of human MEC xenografts, indicating an essential role of Notch signaling in MEC growth in vivo. Finally, since we previously showed that the CRTC1-MAML2-induced autocrine AREG-EGFR signaling was required for the growth and survival of human MEC cells, we reasoned that simultaneous targeting of Notch and EGFR signaling will eliminate cancer stem and non-stem cell populations, likely improving therapeutic efficacy. Indeed, concurrent inhibition of Notch and EGFR signaling via respective DBZ and Erlotinib synergistically blocked the colony and oncosphere formation capacity of human MEC cells in vitro and in vivo. Collectively, our data demonstrate that Notch signaling is a critical regulator for maintaining MEC cancer-initiating cells and that the combination strategy targeting Notch and EGFR signaling is a potentially effective approach for MEC treatment. Citation Format: Wei Ni, Zirong Chen, Xin Zhou, Rongqiang Yang, Frederic Kaye, Lizi Wu. Role of Notch signaling in human mucoepidermoid carcinoma and combined targeting of Notch and EGFR signaling as an anti-cancer strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4837.

PO-430 Natural killer cell-based adoptive immunotherapy is effective in the eradication of chemoresistant stem-like cells in bladder cancer

IntroductionBladder cancer is the most common malignancy of the urinary tract, and one of the leading causes of cancer death in Western countries. Non-muscle invasive bladder cancer (NMIBC) has high risk of recurrence and progression to muscle-invasive forms, which seems to be largely related with the presence of stem-like cell populations known as cancer stem cells (CSCs) that are refractory to conventional therapies. Here, we evaluated the anti-tumoral activity of Natural Killer (NK) cell-based adoptive immunotherapy against competent bladder CSCs in a pre-clinical relevant orthotopic bladder cancer model, using NK cells from healthy donors and NMIBC patients.Material and methodsNK cells collected from healthy donors and NMIBC patients and activated with IL-2/IL-15 were phenotypically characterised and assayed in vitro against CSCs and bulk differentiated bladder cancer cells, using the CD107a degranulation or Cr-51 release assays. CSCs were isolated using the matrigel sphere-forming assay. The in vivo therapeutic efficacy was evaluated in mice bearing a CSC-induced orthotopic model. Animals were treated twice a week by intravesical instillation of activated-NK cells during two weeks. Tumour response was evaluated longitudinally by non-invasive bioluminescence imaging.Results and discussionsNK cells from healthy donors, but not from NMIBC patients, upon IL-2/IL-15 activation, kill indiscriminately both stem-like and differentiated tumour cells, via stress ligands recognition. Moreover, NK cells shifted CSCs towards a more differentiated phenotype rendering them more susceptible to cisplatin, highlighting the benefits of a possible combined therapy. The locoregional administration, via intravesical instillation of healthy activated-NK cells provides an efficient tumour infiltration and subsequent massive decrease in the tumour burden ranging from 80% to complete remission, with high selective cytolytic activity against CSCs.ConclusionAlthough pre-clinical, our results strongly suggest that an immunotherapeutic strategy using allogeneic activated-NK cells is effective against bladder cancer by targeting both stem and non-stem cell populations and should be exploited as a complementary approach in high-risk bladder cancer patients to prevent tumour recurrence and progression.AknowldgementsAstellas European Foundation Uro-Oncology Grant 2013 and FCT (Portugal): PEst-UID/NEU/04539/2013 and FEDER-COMPETE (FCOMP-01–0124-FEDER-028417 and POCI-01–0145-FEDER-007440).

Abstract 3064: Oral cancer stem cells modulate Fusobacterium nucleatum to acquire the capability to induce tumor stemness switch

Abstract Background: Fusobacterium nucleatum is an oral bacteria that contributes to oral carcinogenesis. Its potential role in oral cancer is not known. Fusobacterium nucleatum (henceforth FN) is a gram-negative anaerobe associated with periodontal diseases and also colon cancer. The bacteria modulates the host cell signaling mechanism including the activation of beta-catenin signaling and p38 pathway. The Fap2 is the outer membrane proteins of FN that inhibit lymphocytes and NK cells. The bacteria also directly binds to NKp46 receptor of NK cells. These properties of FN may be exploited by oral CSCs to defend their own niche as a part of niche-defense mechanism against immune system. Hence, it is not surprising that oral CSCs may hijack this specific oral bacterium to enhance tumor stemness. We speculate that oral CSCs may hijack specific bacteria type present in oral mucosa, and enhance their stemness-inducing mechanisms to switch non-CSCs to CSCs. Methods: We have established an in vitro model of oral cancer cells/FN host/pathogen interaction model. Using this model, we wanted to study potential role of the bacteria in cancer stemness. We also used the saliva of subjects with oral cancer to infect oral cancer stem cells and non-stem cancer cells. Results: When the SCC-25 oral cancer cell line was treated with the saliva of subjects with oral cancer, the EpCAM+/ABCG2+ oral CSCs population was increased and showed the presence of FN. The FN recovered from CSCs exhibited high expression of FadA and Fap2, two surface proteins present in FN that can modulate signaling pathways of host cells. Importantly, FN recovered from the infected CSCs induced stemness in the non-stem cancer cell population of SCC-25, suggesting that CSCs may modulate the pathogen. Indeed, our preliminary observation indicates that FN recovered from the FN strain ATCC 25586 infected SCC-25 ABCG2+ cells showed increased expression of FADA and Fap2 as compared to the parental strain ATCC 25586. Conclusion: Our data suggests that CSCs have the capability to reprogram F. nucleatum genome in order to make the bacteria a potent inducer of tumor stemness in non-stem cancer cells. Citation Format: Bidisha Pal, Seema Bhuyan, Debabrat Baishya, Bikul Das. Oral cancer stem cells modulate Fusobacterium nucleatum to acquire the capability to induce tumor stemness switch [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3064.

Abstract 2525: Sema3C promotes glioma stem cell survival and radioresistance

Abstract Introduction: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined. Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed. Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NF-κB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of Plexin receptors were more resistant to radiation, and Sema3C and Plexin receptors were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance. Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index. Citation Format: Jianghong Man, Jocelyn Shoemake, Qiulian Wu, Jennifer Yu. Sema3C promotes glioma stem cell survival and radioresistance. [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 2525.

Overcoming treatment resistance in cancer: Current understanding and tactics

Chemotherapy is the standard treatment for many, if not all, metastatic cancers. While chemotherapy is often capable of inducing cell death in tumors leading to shrinkage of the tumor bulk, many patients suffer from recurrence and ultimately death due to resistance. During the last decade, treatment resistance has attracted great attention followed by some seminal discoveries, including sequential mutations, cancer stem cells, and bidirectional inter-conversion of stem and non-stem cancer cell populations. Nevertheless, the successful treatment of cancer will require a considerable refinement of our knowledge concerning treatment resistance. In doing so, we expect that a more informed and refined approach to treat cancer will be developed and this may improve prognosis of cancer patients. In this review, we will discuss the current knowledge concerning the failure of cancer treatments and the potential approaches to overcome therapeutic resistance.

A Three-Dimensional Organoid Culture System Derived from Human Glioblastomas Recapitulates the Hypoxic Gradients and Cancer Stem Cell Heterogeneity of Tumors Found In Vivo.

Many cancers feature cellular hierarchies that are driven by tumor-initiating cancer stem cells (CSC) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2(+), OLIG2(+), and TLX(+) cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiotherapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displaying histologic features, including single-cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology. Cancer Res; 76(8); 2465-77. ©2016 AACR.

Replicator dynamics of cancer stem cell: Selection in the presence of differentiation and plasticity

The cancer stem cell hypothesis has evolved into one of the most important paradigms in cancer research. According to cancer stem cell hypothesis, somatic mutations in a subpopulation of cells can transform them into cancer stem cells with the unique potential of tumour initiation. Stem cells have the potential to produce lineages of non-stem cell populations (differentiated cells) via a ubiquitous hierarchal division scheme. Differentiation of a stem cell into (partially) differentiated cells can happen either symmetrically or asymmetrically. The selection dynamics of a mutant cancer stem cell should be investigated in the light of a stem cell proliferation hierarchy and presence of a non-stem cell population. By constructing a three-compartment Moran-type model composed of normal stem cells, mutant (cancer) stem cells and differentiated cells, we derive the replicator dynamics of stem cell frequencies where asymmetric differentiation and differentiated cell death rates are included in the model. We determine how these new factors change the conditions for a successful mutant invasion and discuss the variation on the steady state fraction of the population as different model parameters are changed. By including the phenotypic plasticity/dedifferentiation, in which a progenitor/differentiated cell can transform back into a cancer stem cell, we show that the effective fitness of mutant stem cells is not only determined by their proliferation and death rates but also according to their dedifferentiation potential. By numerically solving the model we derive the phase diagram of the advantageous and disadvantageous phases of cancer stem cells in the space of proliferation and dedifferentiation potentials. The result shows that at high enough dedifferentiation rates even a previously disadvantageous mutant can take over the population of normal stem cells. This observation has implications in different areas of cancer research including experimental observations that imply metastatic cancer stem cell types might have lower proliferation potential than other stem cell phenotypes while showing much more phenotypic plasticity and can undergo clonal expansion.

Abstract A45: Semaphorin 3C mediates glioma stem cell tumorigenicity and radioresistance

Abstract Purpose: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined. Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed. Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NFkB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of PlexinD1 receptors were more resistant to radiation, and Sema3C and PlexinD1 were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance. Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index. Citation Format: Jennifer Yu, Jianghong Man, Shoemake Jocelyn, Wu Qiulian, Jeremy Rich, Shideng Bao. Semaphorin 3C mediates glioma stem cell tumorigenicity and radioresistance. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A45.

Regulation of microRNA-200c in cancer stem cells

Stem cells are unspecialized cells that are not only able to differentiate into specialized cell types, but also to self-renew and infinitely reproduce themselves. Through impacting global gene expression, epigenetic regulators, such as microRNAs, play a crucial role in directing cellular programming to govern stem cell states (stemness vs. differentiation) [1-3]. Notably, during tumorigenesis, the generation of cancer stem cells (CSCs), a subset of the cancer cell population with acquired stemness properties associated with normal stem cells, allows the perpetual rise of the bulk of a tumor as the “seed” of the cancer. Earlier published studies have also provided strong evidence that an enlarged CSC population is associated with tumor aggressiveness and recurrence [2, 3]. Therefore, understanding the critical mechanism underlying regulation of cell stemness-differentiation homeostasis to sustain/exhaust the stem cell pool, which is likely to be shared between the normal mammary stem cells and the breast CSC population, holds the key to the development of effective therapeutic interventions to target CSCs and prevent cancer progression and recurrence. The epithelial-mesenchymal transition (EMT) program is key to cancer progression and metastasis and has been linked to generation of CSCs [3]. Our previous work has shown that tumor suppressor p53 plays a role in regulating both EMT and EMT-associated breast CSCs through transcriptional activation of microRNAs involved in regulation of stemness, including microRNA-200c (miR-200c) [4]. Functional (wild-type) p53 transactivates miR-200c through direct binding to the miR-200c promoter, and loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates EMT program, accompanied by increased CSC-like population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties and thereby reverts the mesenchymal and CSC-like phenotype caused by loss/mutation of p53 to a differentiated epithelial cell-like phenotype [4]. Notably, studies have shown that miR-200c is the most down-regulated miRNA in the normal and neoplastic stem cell populations compared with the non-stem cell populations [4, 5]. Furthermore, miR-200c plays an integral and active role in the maintenance of differentiated epithelia to antagonize tumorigenesis by directly targeting key proteins involved in activation of the EMT-CSC traits, such as ZEB1/2 and BMI1 [4, 5], and potentially more targets beyond. Therefore, determining the components and mechanisms underlying the regulation of miR-200c expression likely facilitates the development of strategies that can be used to pharmacologically manipulate miR-200c for effective cancer treatment. It is recognized that obesity is an established risk factor for breast cancer incidence, progression, recurrence, and mortality, and that the dynamic interaction between tumor cells and the local microenvironment plays a crucial role in malignancy development. Interestingly, our recent study shows that in response to increased adiposity, elevated leptin, an adipokine produced by adipocytes, the most abundant cell type surrounding breast epithelia, activates STAT3-G9a to epigenetically silence a cohort of targeted genes and non-coding RNAs involved in regulation of cell differentiation and epithelial homeostasis, including miR-200c [6]. Down-regulation of miR-200c by activated leptin-STAT3-G9a signaling promotes the gain of EMT-CSC traits to expand a subset of highly tumorigenic breast CSCs enriched by the cell surface marker leptin receptor (OBRhi). In contrast, inhibition of STAT3 restores the expression of miR-200c, which in turn converts the CSC phenotype to a differentiated epithelial phenotype. Consistently, STAT3 inhibitor treatment significantly suppresses the CSC-like OBRhi population and abrogates tumor progression of a diet-induced obesity rat model of breast cancer [6]. Together, these studies elucidate critical and sophisticated roles of intrinsic mutation (e.g. loss of p53) and extrinsic influence (e.g. increased adiposity) in directing EMT-MET (mesenchymal epithelial transition) and stemness-differentiation plasticity through regulation of miR-200c expression during tumorigenesis. The results further reveal novel therapeutic implications of re-activation of p53 or inhibition of STAT3 in restoration of miR-200c to eliminate the CSC pool and thereby prevents breast cancer progression/recurrence.

Abstract 1531: Distinct roles of CXCR3 isoforms in promoting breast cancer stem-like cell properties and metastasis

Abstract Growing evidence has demonstrated that the two major isoforms of chemokine receptor CXCR3 (CXCR3-A, CXCR3-B) both contribute to breast cancer pathogenesis and metastasis, however, in different ways. Understanding the relative contribution of each isoform is critical to optimize therapeutic strategies targeting CXCR3. Furthermore, the possible involvement of either CXCR3 isoform in cancer stem-like properties has not been reported. We examined the contribution of each isoform to metastasis using a panel of breast cell lines and a xenograft model of breast cancer; we also explored the role of major CXCR3 isoforms in the behavior of stem-like cells. CXCR3-A is more abundantly expressed than CXCR3-B in both primary human breast cancer tissue and in breast cancer cell lines. On the contrary, immortalized normal MCF-10A cells are CXCR3-B dominant. In basal-like MDA-MB-231 cells, CXCR3 ligand-stimulated proliferation is inhibited by CXCR3-B overexpression, with concurrent reduced activation of ERK1/2 and p38 kinases. Similarly, higher levels of CXCR3-B inhibit migration and invasion in vitro and metastasis in vivo. Accordingly, reduced CXCR3-B expression by gene-silencing enhances lung colonization in the xenograft model. Although exhibiting anti-proliferative and anti-metastatic roles in the non-stem cell population, CXCR3-B supports a cancer stem-like cell phenotype. In mammosphere-forming MDA-MB-231 cells, CXCR3-B is markedly up-regulated and mammosphere-forming capacity is further increased when overexpressing CXCR3-B. Likewise, soft agar growth is promoted by CXCR3-B overexpression. Accordingly, silencing CXCR3-B by shRNA inhibits stem-like properties. In conclusion, both isoforms need to be targeted to inhibit the pro-proliferative and pro-metastasis functions of CXCR3-A and the stem cell-promoting actions of CXCR3-B. Citation Format: Yanchun Li, Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Tyler Kochel, Amy M. Fulton. Distinct roles of CXCR3 isoforms in promoting breast cancer stem-like cell properties and metastasis. [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 1531. doi:10.1158/1538-7445.AM2015-1531

Abstract 3345: Inhibition of STAT3 reduces radiation induced changes in cellular plasticity and sensitizes breast cancer cells to radiation

Abstract Triple negative breast cancer (TNBC) is categorized as a lack of expression of the hormone receptors, estrogen and progesterone, and HER2, and is a highly aggressive form of breast cancer. Due to the receptor status, TNBC is unresponsive to conventional targeted treatment resulting in a poor prognosis and increased risk of recurrence and metastasis. We have observed that TNBC patients have higher amounts of infiltrating leukocytes compared to estrogen receptor positive breast cancer patients. Therefore, inflammation may be a contributing factor in TNBC and inhibition of key inflammatory pathways may aid in sensitizing TNBC cells to treatment. Growing evidence suggests that radioresistant breast cancer stem cells are responsible for the tumor growth, recurrence, and metastasis following treatment. Previous studies have suggested that radiation alters non-stem cell populations to become more stem-like, indicating that radiation may induce changes in cellular plasticity. Cytokine activation of the signal transducer and activator of transcription 3 (STAT3) has been shown to mediate tumor-promoting inflammation and expansion of stem cell populations. Thus, we wanted to determine how inhibition of the STAT3 pathway alters the response of TNBC to radiation. To determine the effect of STAT3 inhibition on breast cancer stem populations, TNBC cells were treated with two different small molecule inhibitors to STAT3, C188-9 or Stattic, to block STAT3 signaling and the enzymatic activity of the cancer stem cell marker, aldehyde dehydrogenase (ALDH), was measured by Aldefluor assay. Treatment with 10 μM C188-9 decreased the ALDH positive (ALDH+) population from 18.3% to 5.2% and treatment with 1 μM Stattic decreased the ALDH+ from 12.2% to 4.9% indicating that inhibition of STAT3 decreases the stem cell population of TNBC. In order to determine the effect of STAT3 inhibition on radiation induced plasticity of breast cancer cells, TNBC cells were isolated into ALDH+ and ALDH negative (ALDH-) populations using flow cytometry and irradiated with 8 Gy of radiation. Following five days of treatment, both populations exhibited an increase in the percentage of ALDH+ cells indicating an increase in stem-like cells following radiation. Treatment with either 10 μM C188-9 or 1 μM Stattic significantly blocked the radiation induced increase in breast cancer stem cells. The combined effect of radiation and STAT3 inhibition on colony formation was also assessed. Exposure to 8 Gy of radiation decreased colony formation; however, inhibition of STAT3 prior to radiation exposure significantly reduced colony formation and thus sensitized the breast cancer cells to radiation treatment. These data suggest that targeting STAT3 could potentially be used to prevent alterations in cellular plasticity induced by radiation, reduce populations of resistant stem-like cells, and improve treatment outcomes. Citation Format: Kimberly M. Arnold, Lynn M. Opdenaker, Daniel Flynn, Jennifer Sims-Mourtada. Inhibition of STAT3 reduces radiation induced changes in cellular plasticity and sensitizes breast cancer cells to radiation. [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 3345. doi:10.1158/1538-7445.AM2015-3345

Immunomic Screening of Cell Surface Molecules on Undifferentiated Human Dental Pulp Stem Cells.

Human adult dental pulp tissue is a source of adult stem cells that have a potential to differentiate into various tissues, although the primary cell suspensions cultured from pulp tissue are mixtures of both stem cell and nonstem cell populations with heterogeneous phenotypes and various differentiation efficiencies. Therefore, cell surface protein markers on dental pulp stem cells are critical for detection and purification of stem cell populations. Yet, little is known about the cell surface molecules that are specifically associated with the undifferentiated and progenitor state of human adult dental pulp stem cells (hDPSCs). Presently, cell surface proteins expressed on hDPSCs were assessed by screening surface molecules specifically expressed on dentinogenic progenitors. Using a decoy immunization strategy, a set of monoclonal antibodies (MAbs) was generated against undifferentiated pulp progenitor cells. Forty-five hybridomas produced MAbs that interacted weakly, if at all, to differentiated pulp cells. Of these, 19 MAbs (18 IgG, 1 IgM) recognized surface molecules on undifferentiated hDPSCs. By multicolor flow cytometric analysis, 40%-60% of newly identified MAb-positive cells were demonstrated to be positive for the CD44 and CD90 mesenchymal markers. When MAb-positive cells were sorted from the heterogeneous pulp cell suspension, mineralization efficiency was increased three to five times compared with MAb-negative cells. The results suggest that the decoy immunization is an efficient method for isolation of MAbs against dentinogenic progenitors. These MAbs will be helpful for identification and enrichment of hDPSCs for efficient dentin regeneration.

Divergent roles of CXCR3 isoforms in promoting cancer stem-like cell survival and metastasis.

There is growing evidence that several chemokine receptors including CXCR3 contribute to metastasis of breast and other cancers, however, in order to target CXCR3 effectively, it is critical to understand the relative contribution of each CXCR3 isoform. Furthermore, the possible contribution of either major CXCR3 isoform (CXCR3-A, CXCR3-B) to cancer stem cell behavior has not been reported. We employed primary invasive ductal carcinomas, a panel of breast cell lines, and a xenograft model of metastatic breast cancer to examine the role of CXCR3 isoforms in the behavior of breast cancer stem-like cells and the contribution of each isoform to metastasis. In primary human breast cancer specimens as well as established breast cancer cell lines, CXCR3-A is more highly expressed than CXCR3-B. Conversely, immortalized normal MCF10A cells express more CXCR3-B relative to CXCR3-A. Overexpression of CXCR3-B in MDA-MB-231 basal-like cells inhibits CXCR3 ligand-stimulated proliferation, which is accompanied by reduced ligand-mediated activation of ERK1/2 and p38 kinases. Likewise, metastatic capacity is reduced in vivo by higher levels of CXCR3-B, and migratory and invasive properties are inhibited in vitro; conversely, silencing of CXCR3-B enhances lung colonization. In contrast to the anti-metastatic and anti-proliferative roles of CXCR3-B in the non-stem cell population, this isoform supports a cancer stem-like cell phenotype. CXCR3-B is markedly elevated in mammosphere-forming parental cells and overexpressing CXCR3-B further enhances mammosphere-forming potential as well as growth in soft agar; stem-like behavior is inhibited in MDA-MB-231shCXCR3-B cells. Targeting of both CXCR3 isoforms may be important to block the stem cell-promoting actions of CXCR3-B, while inhibiting the pro-proliferative and metastasis-promoting functions of CXCR3-A.

Sema3C Promotes the Survival and Tumorigenicity of Glioma Stem Cells through Rac1 Activation

Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the nonstem tumor cell (NSTC) population is not well defined. Here, we show that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/nuclear factor (NF)-κB signaling in an autocrine/paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype invivo. Our study supports the targeting of Sema3C to break this GSC-specific autocrine/paracrine loop in order to improve glioblastoma treatment, potentially with a high therapeutic index.

Prominin-1 (CD133) defines both stem and non-stem cell populations in CNS development and gliomas.

Prominin-1 (CD133) is a commonly used cancer stem cell marker in central nervous system (CNS) tumors including glioblastoma (GBM). Expression of Prom1 in cancer is thought to parallel expression and function in normal stem cells. Using RNA in situ hybridization and antibody tools capable of detecting multiple isoforms of Prom1, we find evidence for two distinct Prom1 cell populations in mouse brain. Prom1 RNA is first expressed in stem/progenitor cells of the ventricular zone in embryonic brain. Conversely, in adult mouse brain Prom1 RNA is low in SVZ/SGZ stem cell zones but high in a rare but widely distributed cell population (Prom1hi). Lineage marker analysis reveals Prom1hi cells are Olig2+Sox2+ glia but Olig1/2 knockout mice lacking oligodendroglia retain Prom1hi cells. Bromodeoxyuridine labeling identifies Prom1hi as slow-dividing distributed progenitors distinct from NG2+Olig2+ oligodendrocyte progenitors. In adult human brain, PROM1 cells are rarely positive for OLIG2, but express astroglial markers GFAP and SOX2. Variability of PROM1 expression levels in human GBM and patient-derived xenografts (PDX) – from no expression to strong, uniform expression – highlights that PROM1 may not always be associated with or restricted to cancer stem cells. TCGA and PDX data show that high expression of PROM1 correlates with poor overall survival. Within proneural subclass tumors, high PROM1 expression correlates inversely with IDH1 (R132H) mutation. These findings support PROM1 as a tumor cell-intrinsic marker related to GBM survival, independent of its stem cell properties, and highlight potentially divergent roles for this protein in normal mouse and human glia.

Cancer Stem Cell Phenotype Is Supported by Secretory Phospholipase A2 in Human Lung Cancer Cells

Lung cancer stem cells (CSCs) are a subpopulation of cells that drive growth, invasiveness, and resistance to therapy. Inflammatory eicosanoids are critical to maintain this malignant subpopulation. Secretory phospholipase A2 group IIa (sPLA2) is an important mediator of the growth and invasive potential of human lung cancer cells and regulates eicosanoid production. We hypothesized that sPLA2 plays a role in the maintenance of lung CSCs. Cancer stem cells from lung adenocarcinoma cell lines H125 and A549 were isolated using aldehyde dehydrogenase activity and flow cytometry. Protein and mRNA levels for sPLA2 were compared between sorted cells using Western blotting and quantitative reverse transcriptase-polymerase chain reaction techniques. Chemical inhibition of sPLA2 and short-hairpin RNA knockdown of sPLA2 were used to evaluate effects on tumorsphere formation. Lung CSCs were isolated in 8.9%±4.1% (mean±SD) and 4.1%±1.6% of H125 and A549 cells respectively. Both sPLA2 protein and mRNA expression were significantly elevated in the CSC subpopulation of H125 (p=0.002) and A549 (p=0.005; n=4). Knockdown of sPLA2 significantly reduced tumorsphere formation in H125 (p=0.026) and A549 (p=0.001; n=3). Chemical inhibition of sPLA2 resulted in dose-dependent reduction in tumorsphere formation in H125 (p=0.003) and A549 (p=0.076; n=3). Lung CSCs express higher levels of sPLA2 than the non-stem cell population. Our findings that viral knockdown and chemical inhibition of sPLA2 reduce tumorsphere formation in lung cancer cells demonstrate for the first time that sPLA2 plays an important role in CSCs. These findings suggest that sPLA2 may be an important therapeutic target for human lung cancer.