Tumor-initiating Properties Research Articles

601. An Optimized Approach to Non-Viral Transfection of Brain Tumor Initiating Cells Derived from Embryonal Tumor with Multilayered Rosettes Using Cationic Reagents

Embryonal tumor with multilayered rosettes (ETMR) is an aggressive type of primitive neuroectodermal brain tumors of the central nervous system that occur primarily in infants and young children. Despite intensive therapy, prognosis is extremely poor with typical survival outlook of <1 year after diagnosis. Resistance to conventional chemo/radiation therapy among ETMR patients has been attributed to the resilience and resurgence of a sub-population of “brain-tumor initiating cells” (BTIC) within the tumor mass. These BTICs are a type of cancer stem cell with the capacity for long-term self-renewal, multi-lineage differentiation, and tumor formation. To better understand the pathogenesis of ETMR and develop effective therapies against this disease, we have previously established and characterized a BTIC cell line (BT183) with molecular signatures and tumor-initiating properties that are hallmarks of ETMR. However, BTICs have so far proven difficult to transfect at high efficiency, making them refractory to genetic manipulation approaches. Here, we describe an optimized approach to transfect BTICs using cationic reagent. We initially tried transfecting BT183 with Lipofectamine-2000 according to manufacturer's suggested protocol, but were not able to get any GFP+ cells. Because Lipofectamine is formulated with a multivalent cationic lipid, one rate-limiting factor could be its susceptibility to serum inactivation. While BTICs are cultured in a defined, serum-free neural stem cell-promoting media, the presence of growth factors and additives (e.g. EGF, bFGF, heparin) may interfere with the maturation and stability of lipoplexes. To test this hypothesis, we incubated lipoplexes in different preparations of the NeuroCult™-XF Proliferation media, each devoid of one or more of the required additives. We then assayed for complex stability using SYBRSafe, an intercalating dye that binds to the minor grooves of free DNA more efficiently than the condensed form of complexed DNA. We noted that the fluorescent intensities of the complexes were significantly higher (1.2x) in media with EGF, bFGF, heparin, suggesting DNA may be partially dissociated in the presence of these additives. We then transfected cells in the different media preparations and saw that only transfection carried out in media devoid of heparin/growth factors had GFP+ cells. While the number of GFP+ cells were too few to quantitate at this stage, we noted that there were more GFP+ at lower seeding densities, but the GFP+ cells were mostly buried among non-transfected cells as neurospheres. We postulated that the tendency for these BTIC to aggregate in a density-dependent manner may interfere with the efficient binding and uptake of lipoplexes; hence methods to prevent them from sticking to each other during transfection might enhance efficiency. We therefore tried attaching them using a number of cell attachment methods (i.e. gelatin, PEI, CELLSTAR). Only CELLSTAR was able to facilitate uniform cell attachment without causing significant cytoxicity. When we eventually transfected BTIC attached to CELLSTAR-treated tissue culture plates in media without heparin or growth factors, we saw a remarkable increase in transfection efficiency with up to 24% of the cells being GFP+. In summary, we showed here that BTICs can be efficiently transfected using cationic reagent by temporarily remove media components that are otherwise inhibitory to transfection and by adapting cell culture format to one that is more conducive to the binding and uptake of DNA complexes. The approaches outlined here may be adapted to other hard-to-transfect suspension cultures such as lymphocytes and leukemic cells.

A convenient and effective strategy for the enrichment of tumor-initiating cell properties in prostate cancer cells.

Stem-like prostate cancer (PrCa) cells, also called PrCa stem cells (PrCSCs) or PrCa tumor-initiating cells (PrTICs), are considered to be involved in the mediation of tumor metastasis and may be responsible for the poor prognosis of PrCa patients. Currently, the methods for PrTIC sorting are mainly based on cell surface marker or side population (SP). However, the rarity of these sorted cells limits the investigation of the molecular mechanisms and therapeutic strategies targeting PrTICs. For PrTIC enrichment, we induced cancer stem cell (CSC) properties in PrCa cells by transducing three defined factors (OCT3/4, SOX2, and KLF4), followed by culture with conventional serum-containing medium. The CSC properties in the transduced cells were evaluated by proliferation, cell cycle, SP assay, drug sensitivity technology, in vivo tumorigenicity, and molecular marker analysis of PrCSCs compared with parental cells and spheroids. After culture with serum-containing medium for 8days, the PrCa cells transduced with the three factors showed significantly enhanced CSC properties in terms of marker gene expression, sphere formation, chemoresistance to docetaxel, and tumorigenicity. The percentage of CD133+/CD44+ cells was ninefold higher in the transduced cell population than in the adherent PC3 cell population (2.25 ± 0.62 vs. 0.25 ± 0.12%, respectively), and the SP increased to 1.22 ± 0.18% in the transduced cell population, but was undetectable in the adherent population. This method can be used to obtain abundant PrTIC material and enables a complete understanding of PrTIC biology and development of novel therapeutic agents targeting PrTICs.

Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells.

Abstract IA07: Systemic regulation of breast cancer progression

Abstract Recurrent disease affects approximately 30% of breast cancer patients but, unfortunately, by the time overt metastases are detected, it is no longer possible to treat breast cancer patients with curative intent. Clinical evidence indicates that in women who are diagnosed with metastatic breast cancer, tumor cells disseminate from the primary tumor prior to surgical resection but can remain indolent for varying periods of time before the patient becomes symptomatic. The reasons why certain disseminated cells remain indolent while others erupt into malignant tumors remain unknown; it is therefore currently difficult to predict with any degree of accuracy which patients will experience disease relapse. Our lab uses various mouse and xenograft models of breast cancer progression to study the early phases of metastatic disease when patients harbor indolent disseminated tumor cells in the periphery at the time of their primary diagnosis. Using triple-negative models, we previously showed that certain tumor systemically support the outgrowth of distant, otherwise indolent disseminated tumors, doing so by secreting cytokines into the host circulation and activating bone marrow hematopoietic cells to become pro-tumorigenic (McAllister, et al., Cell, 2008; Elkabets, et al., JCI, 2011; Castano, et al, Ca Disc, 2013). Using models of luminal breast cancer, we discovered that certain tumors promote the outgrowth of distant tumors by supporting angiogenesis (Kuznetsov, et al., Ca Disc, 2012). More recently, we have begun to identify the properties of disseminated tumor cells that are poised to respond to systemic factors and importantly, the phenotype of those that do not respond to systemic regulation. We have learned that certain systemic factors (cytokines and hematopoietic cells) elicited by primary breast tumors can sustain tumor-initiating properties of disseminated cells. Our findings highlight the systemic environment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from neoadjuvant or adjuvant therapy. Moreover, we hope that this work will pave the way to designing new therapies that can be delivered at a time when what has previously been an inexorable progression to terminal metastatic disease may be prevented. Citation Format: Sandra S. McAllister. Systemic regulation of breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA07.

Kruppel-like Pluripotency Factors as Modulators of Cancer Cell Therapeutic Responses.

Tumor cells inherit from their normal precursors an extensive stress response machinery that is critical for survival in response to challenges including oxidative stress, wounding, and shear stress. Kruppel-like transcription factors, including KLF4 and KLF5, are rarely affected by genetic alteration during tumorigenesis, but compose key components of the stress response machinery in normal and tumor cells and interact with critical survival pathways, including RAS, p53, survivin, and the BCL2 family of cell death regulators. Within tumor cells, KLF4 and KLF5 play key roles in tumor cell fate, regulating cell proliferation, cell survival, and the tumor-initiating properties of cancer stem-like cells. These factors can be preferentially expressed in embryonic stem cells or cancer stem-like cells. Indeed, specific KLFs represent key components of a cross-regulating pluripotency network in embryonic stem cells and induce pluripotency when coexpressed in adult cells with other Yamanaka factors. Suggesting analogies between this pluripotency network and the cancer cell adaptive reprogramming that occurs in response to targeted therapy, recent studies link KLF4 and KLF5 to adaptive prosurvival signaling responses induced by HER2-targeted therapy. We review literature supporting KLFs as shared mechanisms in stress adaptation and cellular reprogramming and address the therapeutic implications. Cancer Res; 76(7); 1677-82. ©2016 AACR.

Sulforaphane targets cancer stemness and tumor initiating properties in oral squamous cell carcinomas via miR-200c induction.

Cancer stem cells (CSCs) are deemed as the driving force of tumorigenesis in oral squamous cell carcinomas (OSCCs). In this study, we investigated the chemotherapeutic effect of sulforaphane, a dietary component from broccoli sprouts, on targeting OSCC-CSCs. The effect of sulforaphane on normal oral epithelial cells (SG) and sphere-forming OSCC-CSCs isolated from SAS and GNM cells was examined. ALDH1 activity and CD44 positivity of OSCC-CSCs with sulforaphane treatment was assessed by flow cytometry analysis. Invitro and invivo tumorigenicity assays of OSCC-CSCs with sulforaphane treatment were presented. We observed that the sulforaphane dose-dependently eliminated the proliferation rate of OSCC-CSCs, whereas the inhibition on SG cells proliferation was limited. Cancer stemness properties including self-renewal, CD44 positivity, and ALDH1 activity were also decreased in OSCC-CSCs with different doses of sulforaphane treatment. Moreover, sulforaphane treatment of OSCC-CSCs decreased the migration, invasion, clonogenicity, and invivo tumorigenicity of xenograghts. Sulforaphane treatment resulted in a dose-dependent increase in the levels of tumor suppressive miR200c. These lines of evidence suggest that sulforaphane can suppress the cancer stemness and tumor-initiating properties in OSCC-CSCs both invitro and invivo.

Evaluation of Breast Cancer Stem Cells and Intratumor Stemness Heterogeneity in Triple-negative Breast Cancer as Prognostic Factors.

Triple-negative breast cancer (TNBC) is a tumor subtype with aggressive behavior and poor clinical outcome for lacking effective therapies. Breast cancer stem cells (BCSCs) have been suggested to have tumor-initiating properties, but it remains unclear whether their presence contributes to the increased aggressiveness and poor prognosis of TNBC. Also, the breast cancers display frequent inter- and intra-tumor heterogeneity, which adds the complexity in diagnosis and predicting prognosis. Here we investigated the clinical relevance and prognostic value of the BCSC markers, CD44+/CD24-, aldehyde dehydrogenase family 1 member A1 (ALDH1A1) and CD133 in 88 TNBC cases. We found that a few patients displayed spatial heterogeneity of the BCSC markers in expression, which was defined as intratumor stemness heterogeneity (ITSH) below. There was no significant correlation between any BCSC marker alone or ITSH and progression-free survival (PFS). Interestingly, the combined BCSC phenotype by CD44+/CD24- and ALDH1A1 was significantly associated with worse PFS (P = 0.009). Further stratification analysis revealed that this combined BCSC phenotype was an independent prognostic factor for PFS in some subgroups. In conclusion, we demonstrated the existence of ITSH in TNBC and found that the ITSH as well as a single BCSC marker was not significantly associated with survival, whereas combing the analysis of BCSC markers could improve prognostic value. Our findings may lead to an improvement of prognostic indicators in TNBC.

Dual-Blocking of PI3K and mTOR Improves Chemotherapeutic Effects on SW620 Human Colorectal Cancer Stem Cells by Inducing Differentiation

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.

Abstract B119: Blocking metabolic stress response with genetic knockdown and selective ligands of sigma-1 receptor in cancer cells

Abstract There is increasing interest in the biological pathways that determine the ability of cancer cells to adapt and survive to metabolic and micro-environmental stress and sustain tumor-initiating and metastatic properties. A better understanding of these pathways may lead to the identification of key nodes and targetable elements for development novel therapeutic strategies for cancer. The sigma-1 receptor (Sig1R) is a ubiquitously expressed membrane-bound protein that acts as ligand-activated molecular chaperone. Sig1R is localized preferentially at the endoplasmic reticulum (ER) and the mitochondria-associated ER membrane (MAM) domains and controls calcium signaling between ER and mitochondria in response to stress signals. Sig1R is often up-regulated in cancer cell lines and human tumors suggesting that it might have a role in tumorigenesis, although detailed functional studies are missing. In this study we examined whether Sig1R sustains proliferation, survival and tumorigenic properties of human cancer cells. Knockdown of Sig1R using small interfering RNA (siRNA) in human prostate and lung cancer cell lines had profound effect on proliferation, clonogenic capability and tumor-sphere formation, indicating reversal of the tumorigenic and stem-like phenotype in absence of Sig1R. Next, in the attempt to discover pharmacological agents that could phenocopy the effects of the genetic knockdown in cancer cells we tested a series of structurally diverse Sig1R ligands selected for high affinity and selectivity for the receptor. We identified various Sig1R ligands that behaved as antagonists of the receptor functions in cancer cells inhibiting clonogenicity and tumor-sphere formation. Effective concentrations of the ligands were in the micromolar range (1-10 μM). At these doses, Sig1R antagonistic ligands almost completely suppressed clonogenic and tumor sphere forming capability of cancer cells. Furthermore, whereas cell proliferation and viability under standard culture conditions were minimally affected by Sig1R ligands, their effects were more pronounced under glucose starvation, a condition that causes metabolic stress in cancer cells. Notably, both Sig1R knockdown and pharmacological antagonists led to impaired mitochondrial function, which was more evident under glucose starvation. Thus, the absence of functional Sig1R reduced mitochondrial activity and adaptability of cancer cells to metabolic stress. These results indicate that the presence of functional Sig1R sustains tumorigenic and stem-like properties by enhancing the metabolic flexibility of cancer cells through the control of ER-mitochondria functions. Highly selective Sig1R antagonists are promising leads for discovering innovative therapeutic strategies and represent effective candidates for pharmacological targeting of stem-like and tumor-initiating cells in human cancers. Citation Format: Gianluca Civenni, Celeste De Monte, Federica Sereni, Sara Allegrini, Roberto Bosotti, Erik Laurini, Bernhard Wunsch, Sabrina Pricl, Giuseppina M. Carbone, Carlo V. Catapano. Blocking metabolic stress response with genetic knockdown and selective ligands of sigma-1 receptor in cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B119.

Minnelide effectively eliminates CD133(+) side population in pancreatic cancer.

BackgroundPancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease hallmarked by limited patient survival. Resistance to chemotherapy, a major cause of treatment failure in PDAC patients, is often attributed to Cancer Stem Cells (CSCs). Pancreatic CSCs are a small subset of quiescent cells within a tumor represented by surface markers like CD133. These cells are responsible not only for tumor recurrence, but also poor prognosis based on their “stem-like” characteristics. At present, conventional therapy is directed towards rapidly dividing PDAC cells and thus fails to target the CSC population.MethodsMIA PaCa-2, S2-013 and AsPC-1 were treated with 12.5 nM triptolide (12 T cells) for 7 days. The surviving cells were recovered briefly in drug-free growth media and then transferred to Cancer Stem cell Media (CSM). As a control, untreated cells were also transferred to CSM media (CSM). The 12 T and CSM cells were tested for stemness properties using RNA and protein markers. Low numbers of CSM and 12 T cells were implanted subcutaneously in athymic nude mice to study their tumorigenic potential. 12 T and CSM cells were sorted for CD133 expression and assayed for their colony forming ability and sphere forming ability. Invasiveness of 12 T cells, CSM and MIA PaCa-2 were compared using Boyden chamber assays.ResultsTreated 12 T cells displayed increased expression of the surface marker CD133 and the drug transporter ABCG2 compared to untreated cells (CSM cells). Both 12 T and CSM cells formed subcutaneous tumors in mice confirming their tumor-initiating properties. When tested for invasion, 12 T cells had increased invasiveness compared to CSM cells. CD133+ cells in both CSM and 12 T showed greater colony and sphere forming ability compared to CD133− cells from each group. Consistent with these data, when injected subcutaneously in mice, CD133− cells from CSM or 12 T did not form any tumors whereas CD133+ cells from both groups showed tumor formation at a very low cell number. Despite pre-exposure to triptolide in 12 T CD133+ cells, treatment of tumors formed by these cells with Minnelide, a triptolide pro-drug, showed significant tumor regression.ConclusionOur results indicated that triptolide enhanced and enriched the “stemness” in the PDAC cell lines at a low dose of 12.5 nM, but also resulted in the regression of tumors derived from these cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0470-6) contains supplementary material, which is available to authorized users.

Tumorsphere as an effective in vitro platform for screening anti-cancer stem cell drugs.

Cancer stem cells (CSCs) are a sub-population of cells within cancer tissues with tumor initiation, drug resistance and metastasis properties. CSCs also have been considered as the main cause of cancer recurrence. Targeting CSCs have been suggested as the key for successful treatment against cancer. Tumorsphere cultivation is based on culturing cancer cells onto ultralow attachment surface in serum-free media under the supplementation with growth factors such as epidermal growth factor and basic fibroblast growth factor. Tumorsphere cultivation is widely used to analyze the self-renewal capability of CSCs and to enrich these cells from bulk cancer cells. This method also provides a reliable platform for screening potential anti-CSC agents. The in vitro anti-proliferation activity of potential agents selected from tumorsphere assay is more translatable into in vivo anti-tumorigenic activity compared with general monolayer culture. Tumorsphere assay can also measure the outcome of clinical trials for potential anti-cancer agents. In addition, tumorsphere assay may be a promising strategy in the innovation of future cancer therapeutica and may help in the screening of anti-cancer small-molecule chemicals.

Ultrasound-targeted microbubble destruction-mediated downregulation of CD133 inhibits epithelial-mesenchymal transition, stemness and migratory ability of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is an aggressive disease with a poor outcome due to the high incidence of metastasis. Cancer stem cells (CSCs) have been identified to be responsible for tumor progression and may be generated by epithelial-mesenchymal transition (EMT) characteristics. CD133 is a specific surface marker for liver cancer stem cells (LCSCs), which is also considered as an important functional factor for tumorigenesis and overall survival in HCC. Ultrasound-targeted microbubble destruction (UTMD) has recently been used as a novel, safe and effective gene transfection technology. The aim of the present study was to elucidate the regulatory mechanism of CD133 and EMT in LCSCs and whether the UTMD-based shRNA delivery system facilitated gene delivery in LCSCs. In the present study, CD133+ cells were isolated from the SMMC-7721 HCC cell line and then transfected with shCD133 mediated by UTMD and liposomes, respectively. Compared to the liposomes group, the UTMD group resulted in significantly improved transfection efficiency. The downregulation of CD133 reversed the EMT program, attenuated self-renewal, proliferation and migration of CD133+ LCSCs and suppressed the growth of CSC tumor xenografts. Additionally, the downregulation of CD133 led to downregulation of the nuclear factor-κB (NF-κB) pathway. The present study demonstrated that CD133 plays a critical role in the regulation of the EMT process, tumor-initiating properties and migratory ability of LCSCs. The UTMD technique targeted for CD133 downregulation may be examined as a potential therapeutic strategy for HCC.

JNK is required for maintaining the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells.

Many studies reveal an association between the acquired chemoresistant phenotype of cancer cells and tumor-initiating cell-like properties. The aim of this study was to determine the impact of c-Jun N-terminal kinase (JNK) on the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells. Two well-established human acquired chemoresistant cancer cell lines K562/A02 and KB/VCR, as well as their respective parental counterparts K562 and KB were tested. The expression of relevant mRNAs and proteins was detected using qRT-PCR and Western blotting, respectively. Sphere formation and self-renewal assays were used to study the tumor-initiating cell-like properties. Soft agar and colony formation assays were used to investigate tumorigenic ability. We observed that suppressing JNK activity by its specific small molecule inhibitor SP600125 or by limiting JNK1/2 expression with JNK1/2 shRNA lentiviruses inhibited the expression of pluripotent stem cell markers such as Oct4, Sox2, and Nanog in KB/VCR cells and K562/A02 cells as well as sphere formation and self-renewal abilities of K562/A02 cells. Additionally, inhibition of JNK activity significantly inhibited the in vitro and in vivo tumor-initiating abilities of KB/VCR cells. Furthermore, our data suggest that blocking JNK activity abundantly inhibited the Hedgehog (Hh) pathway activity, as reflected by reduction of Hedgehog (Hh) pathway target genes Gli1 and ptch1 at the mRNA level as well as Gli-luciferase activity. JNK maintains the tumor-initiating cell-like properties of acquired chemoresistant K562/A02 and KB/VCR cells potentially through activating the Hedgehog pathway. Thus, disruption of tumor-initiating cell-like properties by targeting JNK may be a new approach to combating acquired chemoresistance.

Abstract 2246: Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells

Abstract Among the four main subtypes of breast cancer, basal-like breast cancer (the predominant subtype of triple-negative breast cancer) is of particular concern, due to its high frequency, relative lack of effective therapies and poor prognosis. Ionizing radiation (for example as treatment for other cancers) and cumulative exposure to steroid hormones (as seen in postmenopausal women under hormone replacement therapy) are known risk factors for breast cancer. Because of their tumor-initiating properties, cancer stem cells (CSCs) were proposed to be responsible for relapse and metastasis. In MCF10A non-cancerous basal-like PR-negative cells, progesterone treatment and 1 Gy X-rays generated ALDH+ and CD44+/CD24- CSCs. Here, we report that progesterone activated the Pi3k/Akt pathway via membrane progesterone receptor (mPR). Inhibition of the Pi3k/Akt pathway counteracted the generation of CSCs by progesterone and irradiation. The stimulation of Pi3K/Akt by progesterone resulted in the inactivation of FOXO3 and a downregulation of miR-29 expression. Stabilization of miR-29 expression impeded the generation of CSCs, while its inhibition alone was sufficient to generate CSCs. This study provides a new mechanistic basis for progesterone and radiation-induced breast cancer risk. In addition, the elucidation of new pathways and miRNA regulations involved in CSC generation and maintenance may open the door to potential new strategies for basal cancer primary or adjuvant therapy, by blocking non-CSCs from replenishing the pool of CSCs and by targeting mechanisms responsible for CSC maintenance. Such CSC-focused therapies may be effective at preventing metastasis and relapse. Citation Format: Guillaume Vares, Sei Sai, Bing Wang, Akira Fujimori, Mitsuru Nenoi, Tetsuo Nakajima. Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells. [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 2246. doi:10.1158/1538-7445.AM2015-2246

Plasminogen activator inhibitor-1 as regulator of tumor-initiating cell properties in head and neck cancers.

The existence of tumor-initiating cells (TICs) has been described in head and neck cancers. Plasminogen activator inhibitor-1 (PAI-1) has been demonstrated to act as a prognostic factor in head and neck cancers. Tiplaxtinin (PAI-039), a specific inhibitor of PAI-1, and PAI-1-specific siRNA were used to examine the role of PAI-1 in the self-renewal property of head and neck cancer-TICs by tumorsphere formation. Western blot, real-time polymerase chain reaction, and luciferase-based reporter assay were used to study the effect of PAI-039 in the sex-determining region Y-box 2 (Sox2) expression. PAI-039 suppressed the self-renewal capability of head and neck cancer-TICs derived from head and neck cancer cell lines through the inhibition of Sox2 expression. PAI-039 decreased the activity of the core promoter and the enhancer of the Sox2 gene in head and neck cancer-TICs. Knockdown of PAI-1 expression also inhibited self-renewal and radioresistance properties of head and neck cancer-TICs. The inhibition of PAI-1 by PAI-039 or siRNA could suppress head and neck cancer-TICs within head and neck cancer cell lines through the downregulation of Sox2. © 2015 Wiley Periodicals, Inc. Head Neck 38: E895-E904, 2016.

Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells.

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA-/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR+PSA+, AR+PSA-, AR-PSA-, and AR-PSA+ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA-/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA+ versus PSA-/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA-/lo PCa cells possess distinct epigenetic profiles. As the PSA-/lo PCa cell population is heterogeneous, in the second part, we employ two PSA- (Du145 and PC3) and two PSA+ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.

Abstract P2-06-03: Prostaglandin E receptor EP4 is a therapeutic target in breast cancer stem cells

Abstract There is an urgent need to identify mechanisms underlying the survival of cells expressing cancer stem cell or tumor-initiating properties. The cyclooxygenase-2 (COX-2) pathway is highly expressed in many breast tumors and contributes to poor outcomes. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on the G-protein-coupled receptor EP4. We compared the expression and function of COX-2 and EP4 in mammosphere-forming and bulk populations derived from a panel of human and murine luminal and basal type tumor cells with different metastatic capacities. Expression of both EP4 and COX-2 were markedly increased in mammosphere-forming cells derived from basal and/or metastatic cells relative to the bulk population, but neither COX-2 or EP4 levels were elevated in mammospheres derived from luminal or non-metastatic cells. Breast cancer stem cells, expressing elevated EP4 are correspondingly more sensitive to inhibition with EP4 antagonists both in vitro and in limiting-dilution assays in vivo. Somewhat to our surprise, cancer stem cells remain relatively sensitive to lysis by Natural Killer cells. We have also shown that EP4 blockade protects NK cells from tumor-induced immune suppression. These studies identify EP4 as a potential therapeutic target in the general tumor cell population and show that EP4 targeting may selectively inhibit cells with tumor-initiating or stem cell properties. EP4 antagonists can directly inhibit cancer stem cells and tumor metastasis and indirectly support NK-mediated mechanisms of tumor control. We are delineating the mechanisms by which EP4 and COX-2 are upregulated in cells with stem-like properties. In addition to EP4 and COX-2, STAT3 is upregulated in stem cells. Inhibition of STAT3 reduces mammosphere-forming capacity. Our studies support a mechanism whereby COX-2/EP4 signaling induces STAT3 to support breast cancer stem cell survival by a feed-forward mechanism. Citation Format: Amy Fulton, Namita Kundu, Tyler Kochel, Xinrong Ma, Jocelyn Reader. Prostaglandin E receptor EP4 is a therapeutic target in breast cancer stem cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-03.

Twist1 and Snail link Hedgehog signaling to tumor-initiating cell-like properties and acquired chemoresistance independently of ABC transporters.

The Hedgehog (Hh) signaling pathway has been implicated in acquired chemoresistance. However, it remains unclear whether and how the Hh pathway may maintain the chemoresistant phenotype by controlling the tumor-initiating cell-like properties of acquired chemoresistant cancer cells. In this study, using well-established acquired chemoresistant cancer cells and chemosensitive KB cancer cells with artificially elevated Hh pathway activity, we found that Hh pathway activity may transcriptionally control the expression of twist1 and snail, thereby maintaining the tumor-initiating cell-like properties and consequently the chemoresistant phenotype. Meanwhile, we obtained direct evidence that twist1, which may amplify Hh signaling activity and plays an essential role in limb development, is a direct transcriptional target of Gli, similar to snail. We further observed that the expression of ATP-binding cassette (ABC) transporters was dispensable for the chemoresistance mediated by twist1 and snail. Collectively, these findings demonstrate that twist1, together with snail, links the Hh pathway to the tumor-initiating cell-like properties of chemoresistant cells. This consequently promotes chemoresistance independently of ABC transporters, thereby contributing to future development of strategies for combating chemoresistance through Hh pathway interference. Furthermore, our finding that twist1 is a direct target of the transcription factor Gli improves the interpretation of the association between twist1 and the Hh pathway and the nature of the signaling transduction of the Hh pathway.

Myelin-forming cell-specific cadherin-19 is a marker for minimally infiltrative glioblastoma stem-like cells.

Glioblastoma stem-like cells (GSCs) exhibit stem-like properties, are highly efficient at forming tumor xenografts, and are resistant to many current therapies. Current molecular identifiers of GSCs are scarce and controversial. The authors describe differential cell-surface gene expression profiling to identify GSC-specific markers. Independent human GSC lines were isolated and maintained in standard neural stem cell (NSC) media and were validated for self-renewal, multipotent differentiation, and tumor initiation properties. Candidate upregulated GSCspecific plasma membrane markers were identified through differential Affymetrix U133 Plus 2.0 Array gene expression profiling of GSCs, human NSCs (hNSCs), normal brain tissue, and primary/recurrent glioblastoma multiforme samples. Results were validated by using comparative quantitative reverse transcription polymerase chain reaction and Western blot analysis of GSCs, hNSCs, normal human astrocytes, U87 glioma cell line, and patient-matched serum-cultured glioblastoma multiforme samples. A candidate GSC-specific signature of 19 upregulated known and novel plasma membrane-associated genes was identified. Preferential upregulation of these plasma membrane-linked genes was validated by quantitative polymerase chain reaction. Cadherin-19 (CDH19) protein expression was enhanced in minimally infiltrative GSC lines. Gene expression profiling of GSCs has shown CDH19 to be an exciting new target for drug development and study of GBM tumorigenesis.

Targeting the c-Met/FZD8 signaling axis eliminates patient-derived cancer stem-like cells in head and neck squamous carcinomas.

Cancer stem-like cells (CSC) thought to contribute to head and neck squamous carcinomas (HNSCC) may offer attractive therapeutic targets if a tractable approach can be developed. In this study, we report that silencing c-Met is sufficient to suppress sphere formation, tumor initiation, and metastatic properties of HN-CSC. Pharmacologic inhibition of c-Met with the selective inhibitor PF-2341066 preferentially targeted CSC and synergized with conventional chemotherapy to improve efficacy in a mouse xenograft model of HNSCC, impeding tumor growth and reducing metastasis. Mechanistic investigations showed that CSC elimination was due to downregulation of Wnt/β-catenin signaling in HN-CSC and that the Wnt pathway receptor FZD8 was essential for interactions of c-Met and Wnt/β-catenin signaling in HN-CSC. Notably, ectopic expression of FZD8 rescued the impaired phenotype of HN-CSC where c-Met was inhibited. Furthermore, c-Met upregulated FZD8 through the ERK/c-Fos cascade in HN-CSC. Taken together, our results offer a preclinical proof-of-concept for targeting the c-Met/FZD8 signaling axis as a CSC-directed therapy to improve HNSCC treatment.