Cancer Stem Cell Signaling Research Articles

Slow-Cycling Cancer Stem Cells Regulate Progression and Chemoresistance in Colon Cancer.

Cancer chemoresistance is often attributed to the presence of cancer stem cell (CSC)-like cells, but whether they are homogeneously chemoresistant remains unclear. We previously showed that in colon tumors, a subpopulation of LGR5+ CSC-like cells driven by TCF1 (TCF7), a Wnt-responsive transcription factor, were responsible for tumorigenicity. Here we demonstrate that the tumorigenic subpopulation of mouse LGR5+ cells exists in a slow-cycling state and identify a unique 22-gene signature that characterizes these slow-cycling CSC. Seven of the signature genes are specifically expressed in slow-cycling LGR5+ cells from xenografted human colon tumors and are upregulated in colon cancer clinical specimens. Among these seven, four genes (APCDD1, NOTUM, PROX1, and SP5) are known to be direct Wnt target genes, and PROX1 was expressed in the invasive fronts of colon tumors. PROX1 was activated by TCF1 to induce CDKN1C and maintain a slow-cycling state in colon cancer organoids. Strikingly, PROX1 was required for recurrent growth after chemotherapeutic treatment, suggesting that inhibition of slow-cycling CSC by targeting the TCF1-PROX1-CDKN1C pathway is an effective strategy to combat refractory colon cancer in combination with conventional chemotherapy. SIGNIFICANCE: These findings illustrate the importance of a slow-cycling CSC subpopulation in colon cancer development and chemoresistance, with potential implications for the identified slow-cycling CSC signatures and the TCF1-PROX1-CDKN1C pathway as therapeutic targets.

Targeting SRC Kinase Signaling in Pancreatic Cancer Stem Cells.

The proto-oncogene nonreceptor tyrosine-protein kinase SRC is a member of the SRC family of tyrosine kinases (SFKs), and its activation and overexpression have been shown to play a protumorigenic role in multiple solid cancers, including pancreatic ductal adenocarcinoma (PDAC). PDAC is currently the seventh-leading cause of cancer-related death worldwide, and, by 2030, it is predicted to become the second-leading cause of cancer-related death in the United States. PDAC is characterized by its high lethality (5-year survival of rate of <10%), invasiveness, and chemoresistance, all of which have been shown to be due to the presence of pancreatic cancer stem cells (PaCSCs) within the tumor. Due to the demonstrated overexpression of SRC in PDAC, we set out to determine if SRC kinases are important for PaCSC biology using pharmacological inhibitors of SRC kinases (dasatinib or PP2). Treatment of primary PDAC cultures established from patient-derived xenografts with dasatinib or PP2 reduced the clonogenic, self-renewal, and tumor-initiating capacity of PaCSCs, which we attribute to the downregulation of key signaling factors such as p-FAK, p-ERK1-2, and p-AKT. Therefore, this study not only validates that SRC kinases are relevant and biologically important for PaCSCs but also suggests that inhibitors of SRC kinases may represent a possible future treatment option for PDAC patients, although further studies are still needed.

Differentiation of Cancer Stem Cells by Using Synthetic Small Molecules: Toward New Therapeutic Strategies against Therapy Resistance.

Despite the existing arsenal of anti-cancer drugs, 10 million people die each year worldwide due to cancers; this highlights the need to discover new therapies based on innovative modes of action against these pathologies. Current chemotherapies are based on the use of cytotoxic agents, targeted drugs, monoclonal antibodies or immunotherapies that are able to reduce or stop the proliferation of cancer cells. However, tumor eradication is often hampered by the presence of resistant cells called cancer stem-like cells or cancer stem cells (CSCs). Several strategies have been proposed to specifically target CSCs such as the use of CSC-specific antibodies, small molecules able to target CSC signaling pathways or drugs able to induce CSC differentiation rendering them sensitive to classical chemotherapy. These latter compounds are the focus of the present review, which aims to report recent advances in anticancer-differentiation strategies. This therapeutic approach was shown to be particularly promising for eradicating tumors in which CSCs are the main reason for therapeutic failure. This general view of the chemistry and mechanism of action of compounds inducing the differentiation of CSCs could be particularly useful for a broad range of researchers working in the field of anticancer therapies as the combination of compounds that induce differentiation with classical chemotherapy could represent a successful approach for future therapeutic applications.

Abstract 6691: Delivering intra-tumoral immune modulators and targeting cancer stem cells using recombinant- AAVs

Abstract Solid tumors are composed of heterogeneous cell populations that support rapid growth of cancer cells and suppress immune cell responses. Unleashing the body's own immune response to eradicate cancer cells is one of the most promising ways to treat cancer patients. However, cancer immunotherapy is not without challenges. Some of these include: 1) Phenotypic plasticity and clonal selection in a highly dynamic, heterogeneous and mutable cell population, which leads to de novo and acquired resistance; 2) Immune editing by cancer cells; 3) Expression of immune check point ligands and receptors to suppress immune response locally within the tumor microenvironment, and systemically using exosomes as immune checkpoint carriers; 4) Cross-talk between cancer stem cells and the immune microenvironment; 5) Feasibility of developing personalized treatment strategies, such as cost vs benefit of CAR-T-cell therapy and time constraint, and humanized antibody production; 6) Systemic toxicity of current immunotherapy strategies; To address these challenges, we propose to use a recombinant adeno-associated virus (rAAV) platform to simultaneously enhance tumor immunity and target cancer stem cells by intra-tumoral administration. We express programmed cell death ligand-1 (PD-L1), and B7-H3 (CD276), in combination with a Notch1 decoy. The latter was chosen because: 1) Notch is a crucial signaling component of cancer stem cells (CSCs) maintenance and resistance and 2) Studies have shown that within the tumor, regulatory T-cells (Treg) are activated by OX40 and Notch ligand Jagged1. Blocking Notch signaling would have dual advantages: blocking Notch signaling in CSCs, and preventing activation of immunosuppressive Tregs. Based on current clinical trials and our experimental results, we hypothesized that intra-tumoral injection of rAAV viral particles engineered to express soluble PD-1, B7-H3 and Notch1 decoys in combination with lipopolysaccharide (LPS) is a promising strategy. To test our hypothesis, we used two mouse triple negative breast (TNBC) cancer models: C0321 in the FVB background and M-Wnt in the C57/Bl6 background; both are aggressive, highly metastatic, and similar to human TNBC. Using tumor spheroids in vitro, in the presence of soluble decoys of PD-1, B7-H3 and Notch1 synergistically induced the most effective tumor cell killing, compared to single decoy application. Importantly, intra-tumoral injection of PD-1, B7-H3 and Notch1 decoys in combination with LPS induced necrosis of tumors in vivo in our preliminary findings. Citation Format: Deniz A. Ucar, Giulia Monticone, Fokhrul Hossain, Samarpan Majumder, Dorota Wyczechowska, Matthew J. Dean, Luis Del Valle, Jovanny Zabaleta, Yong Ran, Sudarvili Shanthalingam, Abraham Bert Chabot, Bridgette M. Collins-Burow, Matthew E. Burrow, Barbara Osborne, Todd Eliot Golde, Lucio Miele. Delivering intra-tumoral immune modulators and targeting cancer stem cells using recombinant- AAVs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6691.

Targeting Strategies for Renal Cancer Stem Cell Therapy.

Renal cell carcinoma (RCC) is an intractable genitourinary malignancy that accounts for approximately 4% of adult malignancies. Currently, there is no approved targeted therapy for RCC that has yielded durable remissions, and they remain palliative in intent. Emerging evidence has indicated that renal tumorigenesis and RCC treatment-resistance may originate from renal cancer stem cells (CSCs) with tumor-initiating capacity (CSC hypothesis). A better understanding of the mechanism underlying renal CSCs will help to dissect RCC heterogeneity and drug treatment efficiency, to promote more personalized and targeted therapies. In this review, we summarized the stem cell characteristics of renal CSCs. We outlined the targeting strategies and challenges associated with developing therapies that target renal CSCs angiogenesis, immunosuppression, signaling pathways, surface biomarkers, microRNAs and nanomedicine. In conclusion, CSCs are an important role in renal carcinogenesis and represent a valid target for treatment of RCC patients.

Crosstalk between aldehyde dehydrogenase-1 and chemoresistance in breast cancer: Insights into the role of vitamin D3

AimsAldehyde dehydrogenase-1 (ALDH-1) is considered a signature of breast cancer stem cells and is linked to poor outcomes in breast cancer patients. This study aimed at investigating the effect of vitamin D3 on enhancing the tumor responsiveness to different conventional chemotherapeutic agents, viz., cisplatin, methotrexate, and doxorubicin. Main methodsIn vitro and in vivo experiments were performed using combinations of vitamin D3 and chemotherapeutic agents. Cell cycle analysis and apoptosis assays were performed. Moreover, ALDH-1 expression levels were estimated in cancer cell lines and solid tumors. For solid tumors, tumor volume and histopathological necrotic indices were estimated. Leukocyte presence was also evaluated in tumors using leukocyte common antigen (LCA). Key findingsResults showed a synergistic interaction between vitamin D3 and each of the chemotherapeutic agents on breast cancer cell lines as well as cell cycle arrest at G2/M phase. A decrease in ALDH-1 levels was reported in both breast cancer cells and in tumor tissues. Reductions in tumor volume were also observed in the groups which received the combination therapy. An influence on necrosis rather than apoptosis was also reported, as evidenced by necrotic indices and Bcl-2 expression in tumor sections, respectively. Increased local leukocytes in tumors was also evident, as indicated by increased expression of leukocyte common antigen (LCA). SignificanceOverall, the present study shows that vitamin D3 has an impact on resistance to different chemotherapeutic agents which could be due to the inhibition of ALDH-1, suggesting its use as an adjuvant therapy in cancer patients receiving chemotherapy.

JNK Signaling in Stem Cell Self-Renewal and Differentiation.

C-JUN N-terminal kinases (JNKs), which belong to the mitogen-activated protein kinase (MAPK) family, are evolutionarily conserved kinases that mediate cell responses to various types of extracellular stress insults. They regulate physiological processes such as embryonic development and tissue regeneration, playing roles in cell proliferation and programmed cell death. JNK signaling is also involved in tumorigenesis and progression of several types of malignancies. Recent studies have shown that JNK signaling has crucial roles in regulating the traits of cancer stem cells (CSCs). Here we describe the functions of the JNK signaling pathway in self-renewal and differentiation, which are essential features of various types of stem cells, such as embryonic, induced pluripotent, and adult tissue-specific stem cells. We also review current knowledge of JNK signaling in CSCs and discuss its role in maintaining the CSC phenotype. A better understanding of JNK signaling as an essential regulator of stemness may provide a basis for the development of regenerative medicine and new therapeutic strategies against malignant tumors.

Glioblastoma Multiforme Stem Cell Cycle Arrest by Alkylaminophenol Through the Modulation of EGFR and CSC Signaling Pathways.

Cancer stem cells (CSCs), a small subpopulation of cells existing in the tumor microenvironment promoting cell proliferation and growth. Targeting the stemness of the CSC population would offer a vital therapeutic opportunity. 3,4-Dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), a small synthetic phenol compound, is proposed to play a significant role in controlling the CSC proliferation and survival. We assessed the potential therapeutic effects of THTMP on glioblastoma multiforme (GBM) and its underlying mechanism in various signaling pathways. To fully comprehend the effect of THTMP on the CSCs, CD133+ GBM stem cell (GSC) and CD133- GBM Non-stem cancer cells (NSCC) population from LN229 and SNB19 cell lines was used. Cell cycle arrest, apoptosis assay and transcriptome analysis were performed for individual cell population. THTMP strongly inhibited NSCC and in a subtle way for GSC in a time-dependent manner and inhibit the resistance variants better than that of temozolomide (TMZ). THTMP arrest the CSC cell population at both G1/S and G2/M phase and induce ROS-mediated apoptosis. Gene expression profiling characterize THTMP as an inhibitor of the p53 signaling pathway causing DNA damage and cell cycle arrest in CSC population. We show that the THTMP majorly affects the EGFR and CSC signaling pathways. Specifically, modulation of key genes involved in Wnt, Notch and Hedgehog, revealed the significant role of THTMP in disrupting the CSCs’ stemness and functions. Moreover, THTMP inhibited cell growth, proliferation and metastasis of multiple mesenchymal patient-tissue derived GBM-cell lines. THTMP arrests GBM stem cell cycle through the modulation of EGFR and CSC signaling pathways.

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

Targeting cancer stem cells from a metabolic perspective.

The process of cancer development and progression is driven by distinct subsets of cancer stem cells (CSCs) that contribute the self-renewal capacity as the major impetus to the metastatic dissemination and main impediments in cancer treatment. Given that CSCs are so scarce in the tumor mass, there are debatable points on the metabolic signatures of CSCs. As opposed to differentiated tumor progenies, CSCs display exquisite patterns of metabolism that, depending on the type of cancer, predominately rely on glycolysis, oxidative metabolism of glutamine, fatty acids, or amino acids for ATP production. Metabolic heterogeneity of CSCs, which attributes to differences in type and microenvironment of tumors, confers CSCs to have the plasticity to cope with the endogenous mitochondrial stress and exogenous microenvironment. In essence, CSCs and normal stem cells are like mirror images of each other in terms of metabolism. To achieve reprogramming, CSCs not only need to upregulate their metabolic engine for self-renewal and defense mechanism, but also expedite the antioxidant defense to sustain the redox homeostasis. In the context of these pathways, this review portrays the connection between the metabolic features of CSCs and cancer stemness. Identification of the metabolic features in conferring resistance to anticancer treatment dictated by CSCs can enhance the opportunity to open up a new therapeutic dimension, which might not only improve the effectiveness of cancer therapies but also annihilate the whole tumor without recurrence. Henceforth, we highlight current findings of potential therapeutic targets for the design of alternative strategies to compromise the growth, drug resistance, and metastasis of CSCs by altering their metabolic phenotypes. Perturbing the versatile skills of CSCs by barricading metabolic signaling might bring about plentiful approaches to discover novel therapeutic targets for clinical application in cancer treatments.Impact statementThis minireview highlights the current evidence on the mechanisms of pivotal metabolic pathways that attribute to cancer stem cells (CSCs) with a special focus on developing metabolic strategies of anticancer treatment that can be exploited in preclinical and clinical settings. Specific metabolic inhibitors that can overwhelm the properties of CSCs may impede tumor recurrence and metastasis, and potentially achieve a permanent cure of cancer patients.

Nuclear Factor Erythroid-Derived 2-Like 2-Induced Reductive Stress Favors Self-Renewal of Breast Cancer Stem-Like Cellsviathe FoxO3a-Bmi-1 Axis

Aims: A subpopulation of cancer cells, termed cancer stem cells (CSCs), has stemness properties, such as self-renewal and differentiation, which drive cancer recurrence and tumor resistance. CSCs possess enhanced protection capabilities to maintain reduced intracellular levels of reactive oxygen species (ROS) compared with nonstem-like cancer cells. This study investigated whether reductive stress could regulate self-renewal activity in breast CSCs. Results: We found that manifestation of stemness in breast cancer stem-like cells was associated with an elevated production of reduced glutathione (GSH) maintained by upregulation of glutamate cysteine ligase catalytic subunit (GCLC) and consequently, lowered ROS levels. This was accompanied by upregulation of phospho-AMP-activated protein kinase, FoxO3a, and Bmi-1. Notably, expression of nuclear factor erythroid-derived 2-like 2 (Nrf2) protein was substantially increased in cells undergoing sphere formation. We noticed that expression of Bmi-1 was inhibited after introduction of Nrf2 short interfering RNA into MCF-7 mammosphere cells. Silencing of Nrf2 expression suppressed the xenograft growth of subcutaneously or orthotopically injected human breast cancer cells. Innovation: Association between Nrf2 and self-renewal signaling in CSCs has been reported, but the underlying molecular mechanism remains largely unresolved. This study demonstrates the Nrf2-mediated signaling pathway in maintenance of reductive stress in breast CSCs. Conclusion: Nrf2 overactivation in breast CSCs upregulates GCLC expression and consequently enhances GSH biosynthesis with concurrent reduction in intracellular ROS accumulation, thereby provoking the reductive stress. The consequent upregulation of nuclear FoxO3a and its binding to the promoter of the gene encoding Bmi-1 account for the self-renewal activity of breast cancer stem-like cells and their growth in a xenograft mouse model.

Bioinformatics analysis of regulated MicroRNAs by placental growth factor signaling in cancer stem cells.

Since the effect of placental growth factor (PlGF) on MicroRNAs (miRNAs) at molecular level was remained unknown, the aim was to predict the transcription factors (TFs) and their regulated miRNAs that activated by PlGF and analysis the function, biological processes, and cancer stem cells (CSCs)-related signaling pathways of miRNAs that regulated in PlGF signaling pathway. The aim of this study is to find the TFs that activated by PlGF, we used three online software programs PCViz, PubAngioGen, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Then, the regulatory miRNAs downstream of the TFs were identified by four software TMHD, chipbase, circuits, and transmir databases. Target genes of miRNAs were predicted by three online software program TargetScan, Pictar, and miRanda algorithms. Moreover, Mirwalk database was used to find the validated miRNAs in angiogenesis process. Furthermore, Gene ontology (GO) biological process, GO molecular function, KEGG pathway, BIOCARTA pathway, Panther pathway, and Reactome pathway in Database for Annotation and Visualization and Integrated Discovery tools were used to find the functions and signaling pathways of target genes. Many target genes of miRNAs in PlGF pathway were involved in CSCs-related signaling pathways such as Hedgehog, Wnt/b-catenin, Notch, mTOR, epidermal growth factor EGF, and transforming growth factor-beta signaling pathways. Regulatory miRNAs in PlGF signaling pathway probably promote cell proliferation, migration, tubulogenesis, and metastasize in CSCs. Bioinformatic analysis revealed that regulatory miRNAs and their target genes in PlGF pathway played important roles in the progression of CSCs-related signaling pathways.

YB-1 interplays with ERα to regulate the stemness and differentiation of ER-positive breast cancer stem cells.

Background: Some stemness-associated transcription factors consistently play essential roles in the maintenance of pluripotency or induce the differentiation of cancer stem cells (CSCs). However, the regulatory mechanism of CSC stemness mediated by transcription factors has not been extensively explored. Here, we show that two transcription factors (YB-1 and ERα), which are simultaneously highly expressed in estrogen receptor (ER)-positive CSCs, interact with each other to regulate the stemness and differentiation of ER-positive CSCs.Methods: The expression of YB-1 was examined in ER-positive CSCs and patient specimens. Western blot, real-time PCR, cell viability analysis, tumorsphere formation assay and subcutaneous tumorigenesis assays were used to study the stemness functions of YB-1 and ERα in CSCs. The relationship between YB-1 and ERα in cells was studied by promoter activity analysis, the electrophoretic mobility shift assay (EMSA) and the Co-IP assay. The mechanisms and functional significance of YB-1 in the sensitivity of CSCs to tamoxifen were further investigated with both in vitro and in vivo models.Results: YB-1 was aberrantly upregulated in the cancerous tissue of ER-positive breast cancer patients and in CSCs. Knockdown of YB-1 in ER-positive CSCs significantly inhibited cell stemness and induced differentiation, and the expression of YB-1 could be regulated by estrogen signaling and ERα in ER-positive breast CSCs. The Co-IP results showed that YB-1 interacted directly with ERα specifically in ER-positive non-CSCs and that YB-1 induced ERα degradation by ubiquitination via direct interaction in differentiated cells. Cell differentiation induced by FBS could inhibit YB-1 phosphorylation and promote YB-1 protein transfer from the nucleus to the cytoplasm. Moreover, cell differentiation induced by targeting inhibited the expression of YB-1 in ER-positive CSCs, which increased the sensitivity of cells to tamoxifen in vitro and in vivo.Conclusion: The ERα/YB-1 axis has an important role in the regulation of ER-positive breast cancer stemness. The dephosphorylation of YB-1 and the interaction between YB-1 and ERα may be the switch that initiates the differentiation of ER-positive CSCs. Targeting YB-1 to sensitize ER-positive CSCs to antiestrogen therapy might represent a new therapeutic strategy that warrants further exploration.

Stem cell programs in cancer initiation, progression, and therapy resistance

Over the past few decades, substantial evidence has convincingly revealed the existence of cancer stem cells (CSCs) as a minor subpopulation in cancers, contributing to an aberrantly high degree of cellular heterogeneity within the tumor. CSCs are functionally defined by their abilities of self-renewal and differentiation, often in response to cues from their microenvironment. Biological phenotypes of CSCs are regulated by the integrated transcriptional, post-transcriptional, metabolic, and epigenetic regulatory networks. CSCs contribute to tumor progression, therapeutic resistance, and disease recurrence through their sustained proliferation, invasion into normal tissue, promotion of angiogenesis, evasion of the immune system, and resistance to conventional anticancer therapies. Therefore, elucidation of the molecular mechanisms that drive cancer stem cell maintenance, plasticity, and therapeutic resistance will enhance our ability to improve the effectiveness of targeted therapies for CSCs. In this review, we highlight the key features and mechanisms that regulate CSC function in tumor initiation, progression, and therapy resistance. We discuss factors for CSC therapeutic resistance, such as quiescence, induction of epithelial-to-mesenchymal transition (EMT), and resistance to DNA damage-induced cell death. We evaluate therapeutic approaches for eliminating therapy-resistant CSC subpopulations, including anticancer drugs that target key CSC signaling pathways and cell surface markers, viral therapies, the awakening of quiescent CSCs, and immunotherapy. We also assess the impact of new technologies, such as single-cell sequencing and CRISPR-Cas9 screening, on the investigation of the biological properties of CSCs. Moreover, challenges remain to be addressed in the coming years, including experimental approaches for investigating CSCs and obstacles in therapeutic targeting of CSCs.

Evodiamine Eliminates Colon Cancer Stem Cells via Suppressing Notch and Wnt Signaling.

Evodiamine, an alkaloid contained in traditional Asian herbal medicines that have been used for hundreds years, is interesting due to its cytotoxic effects against many cancers. We examined the effect of evodiamine on the cancer stem cell (CSC) population and the bulk cultured cancer cells (BCC) of colon cancers to examine the double targeting effect. We found that three colon cancer cell lines’ BCC and CSC are effectively targeted by evodiamine. Evodiamine was able to suppress BCC proliferation and induce apoptosis of the cells captured in G2/M phase, as previously reported. However, evodiamine did not cause the accumulation of CSCs at a certain stage of the cell cycle, resulting in the elimination of stemness through an unknown mechanism. By analyzing the expression of 84 genes related to CSCs in two colon cancer cell lines’ CSC, as well as performing further informatics analyses, and quantitative RT-PCR analyses of 24 CSC genes, we found that evodiamine suppressed the expression of the genes that control key signaling pathways of CSC, namely, WNT and NOTCH signaling, to lead CSC elimination. These results suggest that evodiamine should be further developed for targeting both BCCs and CSCs in colon cancers.

Colon cancer stemness as a reversible epigenetic state: Implications for anticancer therapies.

The recent discovery of cancer cell plasticity, i.e. their ability to reprogram into cancer stem cells (CSCs) either naturally or under chemotherapy and/or radiotherapy, has changed, once again, the way we consider cancer treatment. If cancer stemness is a reversible epigenetic state rather than a genetic identity, opportunities will arise for therapeutic strategies that remodel epigenetic landscapes of CSCs. However, the systematic use of DNA methyltransferase and histone deacetylase inhibitors, alone or in combination, in advanced solid tumors including colorectal cancers, regardless of their molecular subtypes, does not seem to be the best strategy. In this review, we first summarize the knowledge researchers have gathered on the epigenetic signatures of CSCs with the difficulty of isolating rare populations of cells. We raise questions about the relevant use of currently available epigenetic inhibitors (epidrugs) while the expression of numerous cancer stem cell markers are often repressed by epigenetic mechanisms. These markers include the three cluster of differentiation CD133, CD44 and CD166 that have been extensively used for the isolation of colon CSCs.and . Finally, we describe current treatment strategies using epidrugs, and we hypothesize that, using correlation tools comparing associations of relevant CSC markers with chromatin modifier expression, we could identify better candidates for epienzyme targeting.

Integrin-EGFR interaction regulates anoikis resistance in colon cancer cells.

Anoikis resistance is an essential property of cancer cells that allow the extra-cellular matrix-detached cells to survive in a suspended state in body fluid in order to metastasize and invade to distant organs. It is known that integrins play an important role in anoikis resistance, but detailed mechanisms are not well understood. Here we report that highly metastatic colon cancer cells showed a higher degree of anoikis resistance than the normal intestinal epithelial cells. These anoikis-resistant cancer cells express high-levels of integrin-α2, β1, and activated EGFR in the anchorage-independent state than the anchorage-dependent state. In contrast, normal intestinal epithelial cells failed to elevate these proteins. Interestingly, a higher co-association of EGFR with integrin-α2β1/-α5β1 was observed on the surface of anoikis-resistant cells. Thus, in the absence of extra-cellular matrix, integrins in association with EGFR activates downstream effectors ERK and AKT and suppress Caspase-3 activation to induce anoikis resistance as was confirmed from the gene-ablation and pharmacological inhibitor studies. Interestingly, these anoikis-resistant cancer cells express high-level of cancer stem cell signatures (CD24, CD44, CD133, EpCAM) and pluripotent stem cell markers (OCT-4, SOX-2, Nanog) as well as drug-resistant pumps (ABCG2, MDR1, MRP1). Altogether, our findings unravel the interplay between integrin-α2β1/-α5β1 and EGFR in anoikisresistance and suggest that the resistant cells are cancer initiating or cancer stem cells, which may serve as a promising target to combat metastasis of cancer.

633P - DACH1 induced stemness of intestinal organoids by directly suppressing bone morphogenetic proteins (BMP) signaling and contributes to intestinal tumorigenesis

BackgroundCrypt base cells are candidates for intestinal epithelial stem cells (ISC). But limited progress has been made concerning the modulation of stem cells. DACH1 is upregulated in LGR5+ ISC cells with stemness maintenance. Hence, we postulated that DACH1 may serve as a putative modulator of intestine stem cells leading to carcinogenesis. MethodsWe established colon normal, adenomas and cancers organoids, which supported stem cell proliferation and undifferentiation in vitro. A stable and efficient culture system for colon epithelial crypts and tumor organoids was use to facilitate investigation of the dynamics and factors affecting colon stem cell niche and carcinogenesis. ResultsWe showed that in the mouse and human intestine, DACH1 was expressed in discrete crypt base. In colorectal cancer, DACH1 expression became widespread, with increased expression and nuclear localization at all stages. ShRNA-mediated inhibition of DACH1 in colon cancer cells inhibited colony formation in vitro and tumor growth in vivo through affecting cancer stem cell signatures. What is more, DACH1 stimulated both colonosphere formation and tumor organoid formation in vitro. Mechanistic characterization indicated that DACH1 recruited and interacts with SMAD4 to suppress BMP signaling pathway. Eventually, DACH1 induced stemness of organoids through BMP-mediated transcriptional increasing of a large number of stem cell signature genes, including Notch1, Msi-2 and Sox9. Here we provided evidence for an essential role of DACH1 in inducing stem cell self-renewal through direct upregulating stem cell signature genes. In detail, DACH1 was shown by microarray analyses to repress BMP induction of Smad signaling, then DACH1 binding to Smad4 was required for repression of Smad signaling ConclusionsIn conclusion, our findings illustrate an essential role of DACH1 signaling in inducing stem cell expansion during intestinal homeostasis and tumor development. This study also provided a mechanistic insight into the function of DACH1 in maintaining stem cell self-renewal via Smad4. Legal entity responsible for the studySan-Jun Cai. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Identification of new cancer stem cell markers and signaling pathways in HER‑2‑positive breast cancer by transcriptome sequencing.

Human epidermal growth factor receptor (HER)-2-positive breast cancer accounts for ~25% of all breast cancer cases, has a high propensity for relapse, metastasis and drug resistance, and is associated with a poor prognosis. Therefore, it is necessary to develop more effective therapeutic targets for the treatment of HER-2-positive breast cancer. CD44+/CD24−/low is currently the most commonly used marker for breast cancer stem cells (CSCs), which are considered the main cause of drug resistance, relapse and metastasis. In the present study, the ratio of CD44+/CD24−/low cells was almost zero in SK-BR-3 cells; however, it was >90% in MDA-MB-231 cells, as determined by flow cytometry. Since SK-BR-3 and MDA-MB-231 cells both exhibit a strong propensity for invasion and migration, it was hypothesized that there may be other markers of CSCs in SK-BR-3 cells. Therefore, transcriptome sequencing was performed for SK-BR-3 and MDA-MB-231 cells. It was observed that several leukocyte differentiation antigens and other CSC markers were significantly more highly expressed in SK-BR-3 cells. Furthermore, the expression of aldehyde dehydrogenase (ALDH)1A3, CD164 and epithelial cell adhesion molecule (EpCAM) was higher in SK-BR-3 cells compared with in other subtypes of breast cell lines, as determined by reverse transcription-polymerase chain reaction and western blot analysis. In addition, the expression levels of ALDH1A3, ALDH3B2 and EpCAM were higher in HER-2-positive breast cancer compared with in paracancerous tissues and other subtypes of breast cancer, as determined by immunohistochemistry. The expression of β-catenin in the Wnt signaling pathway was lower in SK-BR-3 cells compared with in MDA-MB-231 cells, which may be used as a prognostic indicator for breast cancer. These findings may help identify novel CSC markers and therapeutic targets for HER-2-positive breast cancer.

Co-Treatment with Sulforaphane and Nano-Metformin Molecules Accelerates Apoptosis in HER2+ Breast Cancer Cells by Inhibiting Key Molecules

Breast cancer cell lines MCF-10, MCF-7 and BT-474 expressing various levels of HER2 were examined for their response to treatment with sulforaphane (SLFN), metformin (MTFN), Nano-MTFN or combinations. Direct correlation was found between SLFN effect on cell death and HER2 levels. Bioinformatic studies suggested the possibility of additive co-effects on cell fate by SLFN-MTFN co-treatment. This co-treatment specially with SLFN + Nano-MTFN significantly affected the survival of the cells and killed more BT-474 cells than the other two. Cell sensitivity to SLFN-MTFN combination correlated with HER2 expression levels. RT-PCR showed that parallel with cell death, expression of BCL-2, SRC, WNT1, β-catenin and CD44 are diminished, whereas BAX levels are elevated significantly. Cell co-staining indicated that apoptosis percent correlates with cell death following different treatments. We also found that cell death induced by SLFN-MTFN co-treatment is in direct correlation with HER2 levels and increased cell death correlates directly with BAX levels but inversely with levels of cancer stem cell (CSC) signaling genes and CD44. In conclusion, our data indicate that SLFN and MTFN can reduce cancer cell viability via both collaborative and differential effects and suggest that MTFN increases SLFN effectiveness by targeting common molecules/pathways downstream of HER2 and key for CSC signaling.