Hematopoietic Stem Cell Tropism Mediated Targeting of Breast Cancer Stem Cells via Alteration of Tumor Immune-Microenvironment.

The Sixteenth International Symposium of the Hiroshima Cancer Seminar (HCS) Foundation was held on October 22 2006 at the International Conference Center, Hiroshima. The symposium consisted of 10 special lectures and 23 free paper presentations for a poster session. About 230 people were present and actively discussed cancer stem cells. Prior to this symposium, an Open Lecture to the public by HCS and the Japan Society for Dying with Dignity was held on October 21 where Shigehito Yamawaki (Hiroshima University, Hiroshima) and Kazuko Hamanaka (Hamanaka Dermatological Clinic, Hiroshisma) spoke about Psycho-oncology and Breast Cancer to more than 260 people. Eiichi Tahara (Hiroshima Cancer Seminar Foundation), Chairman of the Organizing Committee of the Sixteenth International Symposium, and Chairman of the HCS Foundation, gave an opening address. Tahara introduced a brief background and the purpose of this series of symposia. Since the establishment of the HCS Foundation in 1992, annual international symposia are organized to create an opportunity for basic scientists and clinical researchers to exchange ideas for cancer research, cancer prevention and cancer therapy. This year, the organizing committee planned to explore the important issue of cancer stem cells. Stem cells have a critical role not only in the generation of new populations of normal cells but also in the development of tumors. The balance between self-renewal and differentiation is strictly regulated to maintain normal stem cell pools and to generate the required supply of fully differentiated cells. Recent evidence has suggested that a subset of cancer cells within the tumor, so-called cancer stem cells, may drive the growth and progression of the tumor. Eradication of cancer stem cells may be essential to a cure for cancer. Advances in our knowledge that regulate proliferation, self-renewal, survival and differentiation of cancer stem cells and normal stem cells may shed light on the mechanism that leads to cancer and perhaps improve cancer treatment. The participants will be able to profit by exchanging ideas and learning from the informative presentations and discussions, and will contribute to our understanding of stem cells in relation to cancer development and treatment. Elsa Quintana (University of Michigan, USA) opened the symposium by describing stem cell self-renewal and cancer cell proliferation. Recent advances have highlighted extensive phenotypic and functional similarities between normal stem cells and cancer stem cells. This raises the question of whether it will be possible to develop therapies that eliminate cancer stem cells without eliminating normal stem cells. To address this question, the function of the Pten tumor suppressor was examined. Conditional deletion of Pten in adult hematopoietic cells led to myeloproliferative disease within days and transplantable leukemias within weeks. Pten deletion also promoted hematopoietic stem cell (HSC) proliferation. However, this cell autonomously led to HSC depletion, preventing these cells from stably reconstituting irradiated mice. In contrast to leukemia-initiating cells, HSCs were unable to maintain themselves without Pten. These effects of Pten deficiency were largely mediated by the mammalian target of rapamycin (mTOR) as they could be inhibited by rapamycin. Rapamycin not only depleted leukemia-initiating cells but also restored normal HSC function. Mechanistic differences between the maintenance of normal stem cells and cancer stem cells can thus be targeted to eliminate cancer stem cells without damaging normal stem cells. Her research group is now extending these studies in a variety of directions, testing the cancer stem cell model in various nervous system malignancies, examining the utility of rapamycin in other cancers, and considering clinical trials to test the effectiveness of rapamycin in patients with leukemia. Shin-Ichi Nishikawa (RIKEN Center for Developmental Biology, Kobe) described niche for quiescent cells. Niche has become the most important issue in stem cell biology, but it is still a hypothetical notion that cannot be defined in a better way than the microenvironment supporting stem cell systems. However, the notion of ‘niche’per se is used in more restricted meaning than that of microenvironment, which stands for the special environment involved in the maintenance of most immature stem cells. There may be two types of niche roles; one is to maintain self-renewal and the other is to maintain quiescence. The molecular mechanisms that induce and maintain the quiescent stem cell should be interesting. It is believed that the best model for addressing this issue would be melanocytes, because quiescent stem cells are distinguished from proliferating pools by their location. For the last 5 years, Nishikawa's group have developed methods to isolate quiescent stem cells, defined the molecular difference between quiescent stem cells and proliferating pools, and evaluated the function of some of the molecules in development and the maintenance of melanocyte stem cells. The previous studies were first summarized in this symposium. Quiescent stem cells are induced by multiple steps, suggesting an enormous complexity for the niche. In vivo analysis, however, has difficulty in determining which molecules are indeed sufficient for inducing quiescent stem cells. Hence, a method to culture proliferating melanocytes has been developed. Using this new culture system has started a survey for molecules that convert proliferating pools into quiescent pools. The latest results on candidate molecules that induce quiescent stem cells were presented. As quiescence is also an important factor that confers the resistance of cancer to treatment, Nishikawa also discussed the potential contribution of their melanocyte study to the understanding of cancer stem cells. Lopa Mishra (Georgetown University, USA) gave a speech on TGF-β signaling in stem cells and gastrointestinal cancers. TGF-β family signaling is markedly prominent at the interface between development and cancer in gut epithelial cells. The TGF-β family proteins play key roles in the self-renewal and maintenance of stem cells in their undifferentiated state, whereas changes in TGF-β family signals drive the selection of defined differentiation pathways and their progression of differentiation. When deregulated, changes in the TGF-β family signaling may contribute to impaired differentiation and allow for the development of cancers, thus linking the differentiation of stem cells with the suppression of carcinogenesis. Several TGF-β signaling components are bona fide tumor suppressors with the ability to constrain cell growth and inhibit cancer development at its early stages. Inactivation of at least one of these components (such as the TGF-β receptors, Smad2, or Smad4, and adaptors such as embryonic liver fodrin) occurs in almost all gastrointestinal tumors. For instance, Smad4+/– mice develop gastric tumors and intercrossing of the Smad4+/– genotype into mice with a mutation in the adenomatous polyposis coli tumor suppressor APCΔ716 results in the development of larger and more invasive colorectal tumors than those observed in the presence of the two Smad4 alleles. Moreover, intercrosses between Smad4+/– mice and elf+/– mice result in the development of gastric cancer in 90% of offspring. Interestingly, a third of elf+/– mice develop hepatocellular cancer spontaneously, but not gastric cancer, suggesting that a full dose of Smad4 is sufficient to suppress gastric cancer formation. Bone morphogenetic protein (BMP) signaling also plays an active role in the stem cell compartments of the colon, presumably by suppressing the effects of Wnt signaling and consequently limiting stem cell renewal. Mutations in the BMP receptor BMPR1A, and Smad4 contribute to juvenile intestinal polyposis and Cowden disease, respectively. Furthermore, inactivation of the gene for one of the type-I BMP receptors in mice allows for an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling the human juvenile polyposis syndrome. TGF-β signaling appears to be important for the transition of stem cells to a progenitor and a fully differentiated phenotype in the gastrointestinal system. Accordingly, Smad 2, 3 and 4, adaptors and stem cell proteins regulated by TGF-β may be ivotal for gastrointestinal epithelial cell differentiation. The absence of this drive to normal epithelial differentiation favors the formation of human gastrointestinal carcinoma. Yoshiaki Ito (National University of Singapore, Singapore) reported that RUNX3 links Wnt and TGF-β signaling pathways in gastrointestinal epithelium and referred possible roles of RUNX genes in cancer stem cells. The continued growth and propagation of cancer cells is considered to be dependent on a small subpopulation called cancer stem cells. Cancer stem cells have been studied most intensely in hematopoietic cells and leukemia stem cells (LSC) share the main characteristics of normal hematopoietic stem cells (HSC), namely the self-renewing capacity and multipotency in differentiation, although the latter characteristic is often aberrant in LSC. The RUNX1/AML1 gene encodes a transcription factor essential for the generation of HSC and is frequently targeted in human leukemia. In human RUNX1-related leukemias, the RAS pathway is often concurrently mutated, but the mechanism of the synergism remains elusive. Ito's group recently found that inactivation of Runx1 in mouse bone marrow cells results in an increase in the stem/progenitor cell fraction due to suppression of apoptosis and elevated expression of the polycomb gene Bmi-1, which is important for stem cell self-renewal. Introduction of oncogenic N-RAS into wild-type cells, in contrast, reduced the stem/progenitor cell fraction due to senescence, apoptosis or differentiation. Such detrimental events occurred presumably due to the cellular failsafe program, although hyperproliferation was induced initially by an oncogenic stimulus. Runx1 insufficiency appears to attenuate such failsafe mechanisms, particularly in the stem/progenitor cells, thereby supporting the clonal maintenance of leukemia-initiating cells expressing an activated oncogene. RUNX3 is related to RUNX1 sharing the highly conserved Runt domain. Unlike RUNX1, RUNX3 is ubiquitously expressed in many different types of cells including the epithelial cells of gastrointestinal tract. RUNX3 is a strong candidate for a gastric cancer tumor suppressor functioning downstream of TGF-β pathway. RUNX3 is inactivated very frequently in gastric cancer by epigenetic silencing of the gene and the protein mislocalization. Moreover, the inactivation was also reported in a wide range of other cancer types including colorectal cancer. Our recent study revealed that intestinal epithelial cells of Runx3-null mice show the high proliferation capacity with up-regulated β-catenin/TCF4 activity. RUNX3 was found to negatively regulate the transcriptional activity of β-catenin/TCF4 by inhibiting its DNA-binding activity through the formation of a ternary complex with β-catenin and TCF4. In the mouse model, adenomatous polyps were formed at a similar frequency in Runx3+/– and Apcmin/+ intestine, and in the compound mice having Apcmin/+ and Runx3+/–, adenocarcinomas were induced. It is suggested that the ternary complex composed of RUNX3, β-catenin, and TCF4 is a nodal point to integrate Wnt and TGF-β signaling to regulate intestinal epithelial cells in a complementary fashion. Since Wnt signal is considered to be essential for the maintenance of intestinal stem cells, it is important to study to see if molecular and biological interaction between β-catenin/TCFs and RUNX3 is taking place in intestinal stem cells. Freddy Radtke (Swiss Institute for Experimental Cancer Research, Switzerland) described Notch signaling in self-renewing tissues and cancer. Notch proteins are large single transmembrane receptors, which regulate many cell fate decisions, and differentiation processes during fetal and postnatal developmental. In addition, aberrant Notch signaling has been associated with an oncogenic role in tumorigenesis. Genetic approaches are needed to aim at establishing the role of Notch receptors and/or ligands in self-renewing systems such as the hematopoietic system, the skin or the gut. However, it is not possible by conventional gene targeting as the embryos die during gestation. Therefore, the Cre-loxP system was used to study the physiological role of multiple components of the Notch pathway, in the epidermis of the skin as well as in the intestine of adult mice by an inducible loss of function approach. The role of Notch1 signaling in mammalian skin is not well understood and is mainly based on in vitro studies suggesting that Notch signaling induces differentiation in mammalian skin. Unexpectedly, ablation of the Notch1 gene results first in epidermal hyperplasia followed by the development of skin tumors and facilitated chemical induced skin carcinogenesis, in part explained by reduced p21WAF1/Cip1 levels. Moreover, Notch1 deficiency in the skin results in derepressed β-catenin and shh signaling, which cumulates in the development of basal cell carcinoma-like tumors over time. Thus Notch1 functions as a tumor suppressor gene in mammalian skin. Inducible inactivation of the transcription factor RBP-J (CSL), which mediates Notch signaling of all Notch receptors in the intestine results in the conversion of undifferentiated proliferative crypt cells into postmitotic goblet cells suggesting that Notch signaling is essential for the maintenance of the progenitor/stem cell compartment of the gut. Thus, Notch functions are pleiotropic and context-dependent. Notch can function as: a lineage specifier, oncogene, tumor suppressor and stem cell gate-keeper. Toru Kondo (RIKEN Center for Developmental Biology, Kobe) introduced stem cell-like cells in cancer cell lines. Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Moreover, many tumors and cancer cell lines express stem cell markers, including CD133, CD24, CD44, and adenosine triphosphate-binding cassette transporters, by which the cells pump out a specific fluorescence dye, such as Hoechst33342, as well as anticancer drugs. Therefore, it is possible that cancer cells resemble stem cells or cancers contain stem cell-like cells. Using the flow-cytometry-based side population (SP) analysis that identifies a kind of stem cell, Kondo's group have succeeded in finding SP cells in rat C6 glioma, rat B104 neuroblastoma, human MCF7 breast cancer, and other cancer cell lines. The purified C6 SP cells, but not the other cells, self-renewed and formed tumors, which contain both neuronal marker and glial marker positive cells, when transplanted in vivo, suggesting that C6 SP cells have characteristics of both neural stem cells and cancer. By analyzing the expression profile in both C6 SP and non-SP cells, several factors were identified, which are dominantly expressed in C6 SP cells, and it was found that one of these factors is crucial for the proliferation of C6 cells. Taken together, these findings suggest that SP cells in cancers might be a crucial target for the curable cancer therapy. Kyung-Sun Kang (Seoul National University, Republic of Korea) described cancer stem cells in human breast. Human breast is composed of secretory alveoli connected by a system of branching ducts embedded in connective tissue. These lobulo-alveolar structures were composed of three cell lineages: myoepithelial cells forming the basal layer of ducts and alveoli, ductal epithelial cells that line the lumen of ducts, and alveolar epithelial cells that synthesize milk proteins. These cells proliferate extensively and differentiate during each pregnancy and lactation and undergo apoptosis during mammary involution. Like other organs, human mammary epithelium may be also be derived from a stem cell component. Studies on the properties of human mammary stem cells have been greatly aided by parallel studies on rodent mammary stem cells. Common adult stem cell properties studied on bone marrow, skin, and brain were also applied to study on mammary stem cells. Recently, in vitro culture systems were established that allow for propagation of primary human mammary epithelial stem cells and progenitor cells in an undifferentiated state, based on their ability to proliferate in suspension as spherical structures, which are termed mammospheres. It was described that mammospheres are composed of stem cells and progenitor cells capable of self-renewal and multilineage differentiation and also showed their cell surface marker change when stem/progenitor cells differentiate. Stem cell self-renewal must be regulated to avoid either stem cell loss or hyper proliferation. Misregulation of the self-renewal mechanism might cause carcinogenesis. This theory was called ‘stem cell theory’ in carcinogenesis for a long time. A recent paper by Al-Hajj and colleagues has provided evidence for the existence of stem cells for breast cancer. They demonstrated that small parts of cells in breast tumors have the capacity to proliferate extensively and form new tumors. Masaki Mori (Kyushu University, Beppu) gave a speech on cancer stem cell-like cells in digestive organs. A subset of stem cells, termed side population (SP) cells, has been identified and characterized in several mammalian tissues and cell lines. However, SP cells have never been identified or isolated from gastrointestinal cancers. To isolate SP cells from various human gastrointestinal system cancer cell lines, flow cytometry and the DNA-binding dye Hoechst 33342 were used. Fifteen of 16 cancer cell lines from the gastrointestinal system contained 0.3–2.2% SP cells. Next, differentially expressed genes between SP and non-SP cells of hepatoma HuH7 were analyzed using an oligonucleotide microarray. The expression of GATA6, which is associated with embryonic development and hepatocytic differentiation, was significantly up-regulated in HuH7 SP cells. The expression of ABCG2, ABCB1 and CEACAM6, which are associated with chemoresistance, were also significantly increased in the SP cells. In addition, some epithelial markers and mesenchymal markers were overexpressed in SP cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical staining validated these results, and suggested a multilineage potential for HuH7 SP cells. In hepatoma HuH7 and colorectal SW480 cell lines, SP cells showed evidence for self-renewal, generating both SP and non-SP cells. Finally, chemoresistance to anticancer agents, including Doxorubicin, 5-fluorouracil and Gemcitabine, were compared between HuH7 SP and non-SP cells using an ATP bioluminescence assay. The HuH7 SP cells expressed a higher resistance to Doxorubicin, 5-fluorouracil and gemcitabine compared to non-SP cells. These findings demonstrate that cancers of the gastrointestinal system do contain SP cells that show some characteristics of so-called stem cells. Atsushi Hirao (Kanazawa University, Kanazawa) described the regulation of stem cell self-renewal by tumor-related genes. Recently, it has been shown that key molecules for tumor development play critical roles in the regulation of stem cell self-renewal. Hirao's group demonstrated that ataxia telangiectasia-mutated (ATM), which is a cell cycle checkpoint molecule in response to DNA damage, has an essential role in the self-renewal capacity of hematopoietic stem cells (HSCs). Atm–/– mice over the age of 24 weeks showed progressive bone marrow failure due to a defect in HSC function that was associated with up-regulation of tumor suppressor genes, p16Ink4a and p19Arf, in response to elevated reactive oxygen species (ROS). Treatment of antioxidative agents restored the reconstitutive capacity of ATM–/– HSCs. These data demonstrate that the self-renewal capacity of HSC depends on Atm-mediated inhibition of oxidative stress. In normal HSCs, increasing levels of ROS limits the lifespan in vivo. Elevation of ROS induced HSC-specific phosphorylation of p38 mitogen-activated protein kinase (MAPK) accompanied by the up-regulation of p16Ink4a and p19Arf during serial bone marrow transplantation. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity, indicating that the ROS-p38 MAPK pathway contributes to the exhaustion of the stem cell population. Prolonged treatment with an antioxidant or a p38 MAPK inhibitor extended the lifespan of HSCs in serial transplantation, indicating that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Furthermore, Atm-mediated ROS regulation is shown to be essential for proper DNA recombination, preventing immunodeficiency and lymphomagenesis. Thus, regulation of ROS levels is critical for both tumor development and stem cell self-renewal. Hideki Taniguchi (Yokohama City University, Yokohama) described clonal identification multipotent stem cells in the normal liver and hepatocellular carcinoma using flowcytometric cell sorting. Using flowcytometry combined with in vitro and in vivo single-cell-based assays, hepatic stem cells were prospectively identified with multilineage differentiation potential and self-renewing capability in the developing mouse liver. c-Met + CD49f+/low CD29+ c-Kit- CD45- TER119- cells in fetal liver could be clonally propagated in culture, where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When cells that expanded in vitro were transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes, with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. These data indicate that self-renewing multipotent stem cells are retained in mid-gestational developing liver. Clonally isolated stem cells could be used to reveal the mechanism of cell differentiation in embryonic gut endoderm, and could also provide new insight into therapies for diseases of the digestive system. On the other hand, recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors, as well as putative stem cells in normal solid organs. In this study, side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines were applied in order to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. SP cells demonstrated high proliferative potential and antiapoptotic properties compared with those of non-SP cells. Immunocytochemistry examination revealed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments revealed that only 1 × 103 SP cells were sufficient for tumor formation, while an injection of 1 × 106 non-SP cells did not initiate tumors. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called ‘stemness genes’ were up-regulated in SP cells in HCC cells. In conclusion, Taniguchi proposed that the as SP cells in HCC cells potential and in the cancer stem cell system characterized by was held in the poster with 23 presentations cancer The of a system for hematopoietic stem cells by by for Cancer and Stem University, and of is with of gastric cancer by cell and by (Hiroshima University, Hiroshima) were the best poster Freddy Radtke to the and difference between the normal stem cells and cancer stem cells. For this the to cancer stem cells is It is important to the mechanism of the cancer stem cell for the resistance to in order to cancer.

Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer

Treatment decisions for breast cancer are based on staging and hormone receptor expression and include chemotherapies and endocrine therapy. While effective in many cases, some breast cancers are resistant to therapy, metastasize and recur, leading to eventual death. Higher percentages of tumor-initiating cancer stem cells (CSCs) may contribute to the increased aggressiveness, chemoresistance, and worse outcomes among breast cancer. This may be particularly true in triple-negative breast cancers (TNBCs) which have higher percentages of CSCs and are associated with worse outcomes. In recent years, increasing numbers of long non-coding RNAs (lncRNAs) have been identified as playing an important role in breast cancer progression and some of these have been specifically associated within the CSC populations of breast cancers. LncRNAs are non-protein-coding transcripts greater than 200 nucleotides which can have critical functions in gene expression regulation. The preclinical evidence regarding lncRNA antagonists for the treatment of cancer is promising and therefore, presents a potential novel approach for treating breast cancer and targeting therapy-resistant CSCs within these tumors. Herein, we summarize the lncRNAs that have been identified as functionally relevant in breast CSCs. Furthermore, our review of the literature and analysis of patient datasets has revealed that many of these breast CSC-associated lncRNAs are also enriched in TNBC. Together, this suggests that these lncRNAs may be playing a particularly important role in TNBC. Thus, certain breast cancer-promoting/CSC-associated lncRNAs could be targeted in the treatment of TNBCs and the CSCs within these tumors should be susceptible to anti-lncRNA therapy.

CN06-02: Breast cancer stem cells.

Modeling human hematopoietic cell development from pluripotent stem cells

To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.

Cancer Stem Cell–Directed Therapies: Recent Data From the Laboratory and Clinic

Despite continued improvements in early detection and therapeutic strategies, chemotherapy resistance and relapse remain key obstacles for successful breast cancer therapy. Survivin, a key member of the inhibitor of apoptosis family, is overexpressed in a variety of human cancers, however, its role within cancer stem cells remains elusive. Here we demonstrate that survivin is a key contributor to the drug-resistance in cancer stem cells in a doxorubicin (Dox)-resistant breast cancer cell model (MCF-7/Adr). Through down-regulation of the survivin gene via in vitro siRNA transfection, MCF-7/Adr demonstrated an increased Dox sensitivity (4-fold decrease in IC 50 ). Furthermore, the combined treatment of survivin down-regulation and Dox showed a significant reduction in tumorigenic capacity (from 11.4% to 3.8%) and cancer stem cells frequency (from 20.6% to 9.2%) as determined by the mammosphere formation assay and cancer stem cell surface marker analysis, respectively. To translate this cancer stem cells targeted anticancer strategy into an effective therapeutic, a novel cancer stem cell targeted strategy was developed by developing a fusion of an RNA aptamer to a breast cancer stem cells surface marker EpCAM aptamer with survivin siRNA (EpCAM aptamer-survivin siRNA chimera). Through optimized chemical modifications, an optimal chimera having an 8.3 h half-life in 50% human serum and producing strong dose-dependent in vitro survivin gene silencing efficacy was engineered for further study. To facilitating in vivo imaging and accurate quantification of the chimera without compromising Dicer processing efficiency, FITC and biotin was conjugated to the Chimera 10 at optimized positions. Since Chimera 10 did not show sufficient serum retention in MCF-7/Adr tumour bearing mice, it was further engineered by conjugating a terminal 20 kDa PEG. The resulted PEGylated chimera displayed significant improvements in pharmacokinetic parameters with 5 h and 6 h for elimination half-life and mean residence time, respectively. In addition, an enhanced tumour accumulation was also observed. After confirming the near 80% survivin gene-silencing capacity of the PEGylated chimera in MCF-7/Adr tumours, a 7-day treatment of the xenograft breast cancer was carried out by combining PEGylated chimera and Dox administrations. The chimera-mediated in vivo survivin silencing resulted in significant increase in Dox sensitivity of MCF-7/Adr tumours as evident by the 2-fold decrease in proliferation (via Ki-67 assay) and 10-fold increase in apoptosis (via TUNEL assay) of the tumour underwent combined treatment compared with that treated with Dox alone . In addition the combined chimera and Dox treatment of the xenograft tumours resulted in a the cancer stem cells 5-fold lower tumour forming frequency and a much longer latency in the treated MCF-7/Adr tumour cells compared with those treated with Dox-alone. This cancer stem cells targeting effect was further confirmed by the universal reduction in the expression of stemness proteins, the 2.5-fold and 4.6-fold greater reduction in mammosphere formation efficacy in the primary and secondary sphere assays as well as a ~5-fold reduction of the cancer stem cells population (cell surface marker analysis) after the combinatorial chimera and Dox treatment. The ability of this EpCAM aptamer-survivin siRNA chimera to enter cancer stem cells of the xenograft MCF-7/Adr tumours and reverse their drug resistance to Dox was assessed directly in the purified breast cancer stem cells that had been sorted from MCF-7/Adr tumours treated with the chimera and Dox. Promisingly, the intravenously administered chimera was detected in approximately 89% of breast cancer stem cells and knocked down ~78% survivin , which in turn led to a marked increase in the sensitivity of these purified cancer stem cells to Dox-induced apoptosis. Finally, the combined treatment of survivin silencing and Dox treatment resulted in markedly improved treatment outcome as evident by the greatly extended overall survival of MCF-7/Adr tumour-bearing mice compared to mice treated with Dox alone. Moreover, the aptamer-siRNA chimera did not elicit either an in vitro or in vivo innate immune response, indicating that the suppression of tumour growth by the chimera/Dox co-treatment is not immune mediated and the chimera is probably safe for clinical application. In conclusion, in this project, firstly, we discovered the pivotal role of survivin played in the drug resistance of breast cancer stem cells in a Dox-resistant breast cancer model. Secondly, we developed an aptamer-based breast cancer stem cell-targeted in vivo delivery system for siRNA. With chemical modifications, this aptamer-mediated siRNA delivery system possesses a high therapeutic index, such that combined treatment with low dose of Dox could inhibit stemness, eliminate cancer stem cells, sustain tumour suppression and prolong survival in mice bearing chemoresistant MCF-7/Adr tumours. Thirdly, since EpCAM and survivin gene also highly expressed in some other types of cancers, the anticancer effect of this chimera may be utilised beyond the breast cancer as demonstrated here. Lastly, this work provides a rationale for cancer stem cells targeted siRNA delivery. Theoretically, cancer stem cells can be effectively targeted by RNAi in any cancer as long as there is a critical gene and a reliable surface marker. In conclusion, we have uncovered a crucial role of survivin in chemoresistance in breast tumour initiating cells. Targeting cancer stem cells with an optimized aptamer-mediated siRNA delivery system successfully knocked down the high level expression of survivin in these cells both in vitro and in vivo . Importantly, this approach reversed chemoresistance, transforming a classical chemotherapy drug to one able to act on tumourigenic cells in addition to the bulk tumour cells, which has far reaching implications for future development of efficacious anti-cancer therapies.

Post-treatment enrichment of the tumor initiating CD44+/CD24- breast cancer stem cell (CSC) population is believed to be responsible for breast tumor recurrence and metastasis. Among breast cancer subtypes, triple negative breast cancer (TNBC) particularly is known to have an abundant CSC population, and is characterized by frequent metastatic recurrence. Chloroquine (CQ), an anti-malarial drug, is a lysotropic reagent that inhibits autophagy. Recently, CQ has been shown to reduce the CSC population in various cancers and has been tested in clinical studies. Concurrently, CQ was identified as a potential CSC inhibitor discovered from gene expression signatures of the CD44+/CD24- CSC population. However, little, aside from inhibition of autophagy, is known about the working mechanism of chloroquine in reducing CSCs, particularly in TNBCs. Based on recent recommendations that low doses of CQ be used to limit toxicities in the heart and retina, we investigated how low doses of CQ enhance the antitumor effects of paclitaxel (PTX) and reduce the CSC population. CSC population changes and apoptosis were analyzed using flow cytometry analysis (CSC: CD44+/CD24-; apoptosis: annexin V) and western blot analysis for cleaved caspase 3, and CSC function was measured by mammosphere formation efficiency in SUM159PT, MDA-MB-231, MDA-MB-468, and Hs578T TNBC cell lines. We observed enhanced cytotoxic effects and significant reduction of the CSC population by combined treatment of PTX and CQ compared to single treatment of either PTX (5nM) or CQ (1 or 5 uM) in SUM159, Hs578T, and MDA-MB-231 cells. The enhanced cytotoxicity by co-treatment of CQ and PTX correlated well with inhibition of autophagy, as indicated by cleavage of LC3B, increased expression of p62, and accumulation of autophagosomes. Moreover, the combined treatment inhibited PTX induced STAT3 activation in CSCs and epigenetically regulated gene expression critical in maintenance of CSCs via repression of DNMT1 expression. Finally, we observed enhanced therapeutic efficacy in vivo with the combination of CQ (10-20 mg/kg, daily) and PTX (30 mg/kg, two times per week) when compared to either CQ or PTX alone (p< 0.05). Herein, we demonstrate effective reduction of the CD44+/CD24- CSC population by combined CQ and PTX treatment through autophagy inhibition. Moreover, we found that the low-dose combined treatment of CQ with PTX was able to regulate gene expression by altering DNA methylation, subsequently reducing CSCs in the TNBC cancer cells. Thus, a low dose treatment of CQ along with chemotherapy may be effective in treating TNBC patients, lend further support for in-depth studies on the mechanism of CQ among the subgroups of TNBC. Citation Format: Dong Soon Choi, Elvin Blanco, Sergio M. Granados-Principal, Bhuvanesh Dave, Melissa Landis, Helen Wong, Jenny Chang. Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2013-237

Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization.

Background and purpose: Triple-negative breast cancer (TNBC) is aggressive with poorer prognosis compared to breast cancer that is positive for hormone receptors or HER2. This can be explained further due in part to the existence of breast cancer stem cells (BCSCs). Our lab has found that histone deacetylase (HDAC) inhibitors are effective in the growth suppression of TNBC in vitro and in vivo. To understand the mechanisms involved, we performed RNA-seq analysis for TNBC cells treated with two HDAC inhibitors (LBH589 and Entinostat). Our RNA-seq analysis and studies from other laboratories have found that HDAC inhibitors may modify multiple signaling pathways with undesired effects due to its broad reactivity. To improve the efficacy of HDAC inhibitors in the treatment of TNBCs, we have examined the effects of LBH589 in combination with several drugs. Among them, Salinomycin works effectively with LBH589. It has been used as an antibiotic for farm animals, but has been identified from a screen of a large library of chemicals to target BCSCs (Gupta et al., Cell, 2009). The purpose of this study is to examine the synergistic effect between LBH589 and Salinomycin in the anticipation of their clinical utility and to evaluate a new target therapy to treat TNBC more efficiently and completely. Material and method: Two TNBC cell lines (HCC1937, MDA-MB-231) were used as models to examine the combined effects of Salinomycin and LBH589. Cell proliferation studies were performed through 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. To evaluate the effects on BCSC population, mammosphere assay was used for estimating self-renewal capacity and ALDEFLUOR analysis was used for monitoring the distribution of BCSCs in treated samples. For tumor induction experiments, ALDH+ cells were injected into NSG mice. As a therapeutic study, single or combined use of LBH589 (10mg/kg) and Salinomycin (5mg/kg) were peritoneally injected into non-obese diabetic scid gamma (NSG) mice 3 days a week. Tumor volume, body weight and food intake were checked weekly. For mechanistic analysis, qPCR, Western blotting and IHC were performed for gene and protein expression using tumors from mice from different treatments. Results: Salinomycin and LBH589 worked synergistically in the suppression of the proliferation of TNBCs (IC50 was 68.8nM and 13.1nM, respectively). Similarly, both drugs inhibited mammosphere formation and ALDH positive population in a synergistic manner. The combination of LBH589 (16nM) with Salinomycin (60nM) reduced 60% of mammosphere formation and 94% of ALDH positive population compared with DMSO treatment. In a mouse model, the combination of LBH589 and Salinomycin had an inhibitory effect on tumor growth compared with the control group and the groups treated with single drug. In all groups, no side effect was seen. The results from mechanistic studies showed that the combination of LBH589 and Salinomycin regulated the Wnt/b-catenin pathway. Discussion: We have evidence that the combination of LBH589 and Salinomycin has a synergistic effect on TNBC through Wnt pathway and suggested this combination could potentially be a new therapeutic strategy for exploring targeting therapy in TNBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-06.

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

Changes in the frequencies of human hematopoietic stem and progenitor cells with age and site

Background: Triple-negative breast cancer (TNBC) is a destructive form of breast malignancy that deficiencies the opportunities of targeted therapeutics. Developing evidence proposes that breast cancer stem cells (BCSCs), considered the main reason for poor prognosis as they induce resistance, metastasis, and relapse. Aim of the Study: This study aims at examining the efficacy of Doxorubicin, Cyclophosphamide, Salinomycin, Digoxin, and Nano-silica encapsulated copra venom in eradicating BCSCs and managing TNBC. Methods: We performed our study using the TNBCs cell line (MDA-MB-231). Our study involved 5 groups with 5 different interventions; Untreated cells, cells treated by Doxorubicin 3.5 µM only, cells treated by a combination of Salinomycin 15 µM and Doxorubicin 3.5 µM, cells treated by a combination of Digoxin 120 nM and 1 mM Cyclophosphamide and cells treated by a combination of (Naja Haje) venom 3.5 µM and Salinomycin 15 µM. Results: Outcomes of our study demonstrated that the involved therapeutics inhibited the growth of the MDA-MB-231 cell line in a significant way by different efficacies and potencies. Cells treated with the combination of doxorubicin and salinomycin were significantly inhibited at a higher level than those treated with doxorubicin only Digoxin and Cyclophosphamide combination exhibited a lower efficacy than the previous combination in inhibiting the growth of cancer cells. Naja haje venom and Salinomycin combination showed the most significant inhibition of MDA-MB-231 cell line growth. Conclusion: The current study confirms the potency of these novel approaches in the management and the eradication of triple-negative breast cancer.

Treatment of triple-negative breast cancer (TNBC) remains challenging. Cancer stem cells (CSCs) are the most intractable subpopulation of TNBC cells, which has associated with a high risk of relapse and poor prognosis. However, eradication of CSCs continues to be difficult. Here, we integrated the multiomics data of our large TNBC cohort (n=360) to identify vital markers of CSCs. We discovered that EMSY, inducing a BRCAness phenotype, was preferentially expressed in breast CSCs, promoted the enrichment of ALDH+ cells and was positively correlated with poor relapse-free survival. Mechanistically, EMSY competitively bound to the Jmjc domain, which was critical for the enzyme activity of the KDM5B-specific H3K4 demethylase to reshape methionine metabolism and promote CSC self-renewal and tumorigenesis in an H3K4 methylation-dependent manner. Moreover, EMSY accumulation in TNBC cells sensitized them to PARP inhibitors against bulk cells and methionine deprivation against CSCs. These findings indicate that clinically relevant eradication of CSCs could be achieved with a strategy that targets CSC-specific vulnerabilities in amino acid metabolism. EMSY was highly expressed in BCSCs and positively correlated with TNBC malignancy a. Heatmap with mRNA expression characteristics of non-CSC and CSC subtypes. The top 2,000 most differentially expressed genes used for clustering are plotted. Samples are also annotated on top by the FUSCC subtypes5. b. Volcano plot showing genes targeted by a siRNA inducing a significant change (Fold change (FC) represented the proportion of ALDH+ cells compared to the control)34. c. Overlap analysis of the upregulated genes in CSC subtype (Gene cluster in A) and the genes via siRNA (Gene cluster in B) inducing a significant reduction of the ALDH+ cell ratio. d. The mRNA expression of EMSY in non-CSC and CSC TNBC transcriptomic subtypes (n = 360, Mann-Whitney’s test). Each boxplot showed the median and 95% confidence intervals (95% CI). e. The CNV frequency of EMSY in non-CSC and CSC subtypes. f, g. EMSY mRNA expression of samples with different EMSY copy number in FUSCC (n=302) and TCGA (n=71) TNBC cohorts. h, i. Immunohistochemistry analysis of EMSY expression in specimens of patients between non-CSC (n=45) and CSC (n=20) subtypes. Representative images (h) and the graph of H Scores (i, mean ± SEM, Mann-Whitney’s test) were shown. Scale bar, 4μm (left), 20μm (right). j, k. Kaplan–Meier RFS plots of EMSY in FUSCC (n=360) and GSE234669 (n=442) TNBC (https://kmplot.com/analysis/). Accumulation of EMSY enhanced CSC self-renewal and tumorigenesis a. The mRNA (left) and protein (right) expression of EMSY in FACS sorted ALDH+ and ALDH− cells. b-d. ALDH activity detected by ALDEFLUOR assay and shown as mean ± SEM (n=3/group, one-way ANOVA’s test) in EMSY-knockdown cells (b, c) and EMSY-overexpressing cells (d). e, f. Self-renewal ability determined by primary mammosphere formation (e, n=6 biological independent experiments, one-way ANOVA’s test) and secondary mammosphere formation (f, n=6 biological independent experiments, one-way ANOVA’s test) in EMSY-knockdown SUM149 and MDA-MB-453 cells. Scale bar, 200 μm. g. Self-renewal ability determined by primary mammosphere formation and secondary mammosphere formation in EMSY-overexpressing SUM159 (n=6 biological independent experiments, one-way ANOVA’s test). Scale bar, 200 μm. h-l. EMSY-knockdown SUM149 were injected into mammary fat pads with extreme limiting dilutions (5 x 104, 5 x 103 cells/fat pad). Tumor growth curves (n=5/group, two-way ANOVA’s test) (h, i) were shown. Tumor cell ALDH activity was determined by ALDEFLUOR assay and the bar plot was shown as mean ± SEM (n=5/group, one-way ANOVA test) (j). Frequency of breast CSC was calculated based on the positive tumor sites per group by ELDA (ELDA: Limiting Dilution Analysis for stem cell research (wehi.edu.au)) (k, l). Dairy methionine deprivation, alone or in combination with PARPi, as a therapeutic strategy for EMSY-amplification tumors. a. Immunohistochemistry analyses of EMSY expression in specimens of patients and representative images were shown. b. Patient-derived organoid (PDO) models were performed with fresh isolated primary tissues derived from four independent TNBC patients according to the mRNA level of EMSY. PDO was cultured with fresh medium with (Complete) or without methionine (methionine deprivation, MD) and tumor sphere numbers were determined (n=6/group, two-way ANOVA test). c-e. Effect of MD and olaparib on CSCs and tumor progression. Mice bearing patient-derived xenograft model (PDX, FD-009) were treated with PARPi (50mg/kg, i.p., once every day), dairy MD, and their combination. Tumor volume (c), tumor cell ALDH activity (d) and absolute ALDH+ cancer cells in tumor tissue (e) were assessed (n=5/group, one/two-way ANOVA test). f. Single breast cancer cells from the primary tumors were implanted for a second tumor formation in a limited dilution assay (1000 and 10,000 cells/fat pad); frequency of breast CSC was calculated based on the positive tumor sites per group by ELDA (ELDA: Limiting Dilution Analysis for stem cell research (wehi.edu.au)). Citation Format: Cui-Cui Liu, Ke-da Yu. EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-07.