Cancer Stem Cells Sphere Research Articles

Caffeic acid inhibits the tumorigenicity of triple-negative breast cancer cells through the FOXO1/FIS pathway

Triple-negative breast cancer (TNBC) still one of the most challenging sub-type in breast cancer clinical. Caffeic acid (CA) derived from effective components of traditional Chinese herbal medicine has been show potential against TNBCs. Our research has found that CA can inhibit the proliferation of TNBC cells while also suppressing the size of cancer stem cell spheres. Additionally, it reduces reactive oxygen species (ROS) levels and disruption of mitochondrial membrane potential. Simultaneously, CA influences the stemness of TNBC cells by reducing the expression of the stem cell marker protein CD44. Furthermore, we have observed that CA can modulate the FOXO1/FIS signaling pathway, disrupting mitochondrial function, inducing mitochondrial autophagy, and exerting anti-tumor activity. Additionally, changes in the immune microenvironment were detected using a mass cytometer, we found that CA can induce M1 polarization of macrophages, enhancing anti-tumor immune responses to exert anti-tumor activity. In summary, CA can be considered as a lead compound for further research in targeting TNBC.

Abstract 3583: Enzalutamide and flutamide induce cytotoxicity in desmoplastic small round cell tumor independent of the androgen receptor

Abstract Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, pediatric cancer caused by the EWSR1::WT1 fusion protein. DSRCT predominantly occurs in males, which comprise 80-90% of the patient population. While the reason for the male prevalence remains unknown, one hypothesis is that the androgen receptor (AR), when stimulated by testosterone in males, plays a critical role in DSRCT tumor growth. As a rare pediatric cancer, DSRCT model systems including cell lines and patient-derived xenografts are scarce. Further, DSRCT cell lines have been thought to seed xenograft tumors poorly, with studies commonly using 5-10 million cells for one xenograft. Using a recently established DSRCT cancer stem cell (CSC) model, we sought to establish DSRCT xenografts from fewer cells to enable large-scale xenograft feasibility and reliability. To our surprise, we were able to consistently seed tumors with as few as 100 cells, not only from CSC spheres but also from standard adherent cells. We hypothesized that mouse sex may explain our ability to seed xenografts with so few cells, as many previous studies have utilized female mice. This observation led us to comprehensively investigate the role of the AR in DSRCT. Here, we demonstrate that AR is highly expressed in DSRCT relative to other fusion-driven sarcomas and that the AR antagonists - enzalutamide and flutamide - reduce DSRCT growth. We further elucidate a novel interaction between AR and EWSR1::WT1, which may explain their co-occupancy in a subset of regulatory genomic regions. However, despite these findings, which suggest an essential role for AR in DSRCT, we find that DSRCT cell lines can form xenografts in female mice at the same rate as male mice and AR depletion does not significantly alter DSRCT growth in vitro. Further, we demonstrate that AR antagonists remain effective in DSRCT cells depleted of AR, establishing an AR-independent mechanism of action. Intriguingly, high-dose enzalutamide treatment reduces EWSR1::WT1 oncoprotein expression and could explain its toxicity. DSRCT cells express high levels of NR3 nuclear receptors, including the glucocorticoid receptor (GR). Enzalutamide was shown to act on GR in AR-negative prostate cancer cell lines, which could possibly explain its effect on DSRCT independent of AR. Further examination of this mechanism may provide promising therapeutic implications for DSRCT and other fusion proteins driven by the EWSR1 promoter. Citation Format: Justin W. Magrath, Ilon N. Goldberg, Danh D. Truong, Alifinai B. Hartono, Shruthi Sanjitha Sampath, Chandler E. Jackson, Anushka Ghosh, Derrick L. Cardin, Haitao Zhang, Joseph A. Ludiwg, Sean B. Lee. Enzalutamide and flutamide induce cytotoxicity in desmoplastic small round cell tumor independent of the androgen receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3583.

Anti-ENO1 antibody combined with metformin against tumor resistance: a novel antibody-based platform.

Antibody-based platforms (i.e., ADC) have emerged as one of the most encouraging tools for the cancer resistance caused by cancer stem cells (CSCs) enrichment. Our study might provide a promising therapeutic direction against drug resistance and serve as a potential precursor platform for screening ADC. The cell migration, invasion, drug resistance, and self-renewal were assessed by the cell invasion and migration assay, wound healing assay, CCK-8 assay, colony formation assay, and sphere formation assay, respectively. The expression profiles of CSCs (ALDH+ and CD44+) subpopulations were screened by flow cytometry. The western blot and cell immunofluorescence assay were used to evaluate pathway-related protein expression in both anti-ENO1 antibody, MET combined with DPP/CTX-treated CSCs. In the present study, western blot and flow cytometry verified that anti-ENO1 antibody target the CD44+ subpopulation by inhibiting the PI3K/AKT pathway, while metformin might target the ALDH+ subpopulation through activation of the AMPK pathway and thus reverse drug resistance to varying degrees. Subsequently, in vitro investigation indicated that anti-ENO1 antibody, metformin combined with cisplatin/cetuximab could simultaneously target ALDH+ and CD44+ subpopulations. The combination also inhibited the CSCs proliferation, migration, invasion, and sphere formation; which may result in overcoming the drug resistance. Then, molecular mechanism exploration verified that the anti-ENO1 antibody, metformin combined with cisplatin/cetuximab inhibited the Wnt/β-catenin signaling. The study preliminarily revealed anti-ENO1 antibody combined with metformin could overcome drug resistance against CSCs by inhibiting the Wnt//β-catenin pathway and might serve as a potential precursor platform for screening ADC. More importantly, it is reasonably believed that antibody-based drug combination therapy might function as an encouraging tool for oncotherapy.

β-catenin inhibitor ICG-001 suppress cell cycle progression and induce autophagy in endometrial cancer cells.

The incidence of endometrial cancer has been rising in recent years. Gene mutation and high protein expression of β-catenin are commonly detected in endometrioid endometrial cancer. ICG-001 is a β-catenin inhibitor via blocking the complex formation of β-catenin and cAMP response element-binding protein (CREB)-binding protein (CBP). This study aims to investigate the effect of ICG-001 on endometrial cancer inhibition. First, endometrial carcinoma patient-derived xenograft (PDX)-derived organoids and primary cells were used to verify the inhibiting ability of ICG-001 on endometrial cancer. Furthermore, endometrial cancer cell lines were used to investigate the anticancer mechanism of ICG-001. Using MTT assay and tumor spheroid formation assay, ICG-001 significantly reduced the cell viability of HEC-59 and HEC-1A cells. ICG-001 enhanced cisplatin-mediated cytotoxicity. ICG-001 decreased cancer stem cell sphere formation. ICG-001 decreased the protein expressions of CD44, hexokinase 2 (HK2), and cyclin A. ICG-001 lowered the cell cycle progression by flow cytometer analysis. Autophagy, but no apoptosis, was activated by ICG-001 in endometrial cancer cells. Autophagy inhibition by ATG5 silencing enhanced ICG-001-mediated suppression of cell viability, tumor spheroid formation, and protein expression of cyclin A and CD44. This study clarified the mechanism and revealed the clinical potential of ICG-001 against endometrial cancer.

Effect of microserum environment stimulation on extraction and biological function of colorectal cancer stem cells

Background3D cancer stem cell (CSC) cultures are widely used as in vitro tumor models. In this study, we determined the effects of enriching HCT116 tumor spheres initially cultured in serum-free medium with different concentrations of serum, focusing on the effect of microserum environment stimulation on extraction and biological function of colorectal cancer stem cells (CCSCs).MethodsCCSCs were enriched in standard serum-free medium and serum-free medium with different concentrations of serum for 1 week. The expression of CSC-associated markers in CCSCs, and the presence and relative proportion of CSCs (CD133/CD44 cell sorting) were then determined to elucidate the effect of the microserum environment on the preservation of CSC-related features. Further, the tumorigenic capacity of CCSCs was evaluated in an immunodeficiency mouse model.ResultsOur data indicated that a significantly greater number of spheres with a greater size range and high viability without drastic alteration in biological and structural features, which maintained self‐renewal potential after sequential passages were formed after serum supplementation. Real-time analysis showed that both serum spheres and serum-free spheres displayed similar expression patterns for key stemness genes. Serum spheres showed higher expression of the CSC surface markers CD133 and CD44 than did CSCs spheres cultured in serum-free medium. Adherent cultures in complete medium could adapt to the serum-containing microenvironment faster and showed higher proliferation ability. The addition of serum induced EMT and promoted the migration and invasion of serum globular cells. Compared with serum-free cells and adherent cells, serum spheres showed higher tumor initiation ability.ConclusionsMicroserum environment stimulation could be an effective strategy for reliable enrichment of intact CCSCs, and a more efficient CSC enrichment method.

Targeting mitochondrial dynamics by AZD5363 in triple-negative breast cancer MDA-MB-231 cell–derived spheres

Breast cancer stem cells (BCSCs) have been suggested to contribute to chemotherapeutic resistance and disease relapse in breast cancer. Thus, BCSCs represent a promising target in developing novel breast cancer treatment strategies. Mitochondrial dynamics in BCSCs were recently highlighted as an available approach for targeting BCSCs. In this study, a three-dimensional (3D) cultured breast cancer stem cell spheres model was constructed. Mitochondrial dynamics and functions were analyzed by flow cytometry and confocal microscopy. We have demonstrated that the protein levels of FIS 1 and Mitofusin 1 were significantly increased in BCSCs. Moreover, Capivasertib (AZD5363) administration could suppress Mitofusin1 expression in BCSCs. Our use of MitoTracker Orange and annexin V double-staining assay suggested that AZD5363 could induce apoptosis in BCSCs. The sensitivity of stem cell spheres to doxorubicin was investigated by CCK8 assay, and our results indicated that AZD5363 could re-sensitize BCSCs to Doxo. Flow cytometry analysis identified doxo-induced CD44 and CD133 expression in BCSCs could be suppressed by AZD5363. In combination with AZD536, doxo-induced apoptosis in the BCSCs was significantly increased. In conclusion, our study explored, for the first time, that AZD5363 could target mitochondrial dynamics in 3D cultured stem cell spheres (BCSCs) by regulating Mitofusin.

PARP-1 Expression Influences Cancer Stem Cell Phenotype in Colorectal Cancer Depending on p53

Poly(ADP-ribose) polymerase-1 (PARP-1) is a protein involved in multiple physiological processes. Elevated PARP-1 expression has been found in several tumours, being associated with stemness and tumorigenesis. In colorectal cancer (CRC), some controversy among studies has been described. In this study, we analysed the expression of PARP-1 and cancer stem cell (CSC) markers in CRC patients with different p53 status. In addition, we used an in vitro model to evaluate the influence of PARP-1 in CSC phenotype regarding p53. In CRC patients, PARP-1 expression correlated with the differentiation grade, but this association was only maintained for tumours harbouring wild-type p53. Additionally, in those tumours, PARP-1 and CSC markers were positively correlated. In mutated p53 tumours, no associations were found, but PARP-1 was an independent factor for survival. According to our in vitro model, PARP-1 regulates CSC phenotype depending on p53 status. PARP-1 overexpression in a wild type p53 context increases CSC markers and sphere forming ability. By contrast, those features were reduced in mutated p53 cells. These results could implicate that patients with elevated PARP-1 expression and wild type p53 could benefit from PARP-1 inhibition therapies, meanwhile it could have adverse effects for those carrying mutated p53 tumours.

Development and Verification of a Prognostic Stemness-Related Gene Signature in Triple-Negative Breast Cancer

Background. It is well known that cancer stem cells can induce cancer metastasis, which causes the majority of cancer-related death, especially in triple-negative breast cancer (TNBC). TNBC features a high metastatic rate and low metastasis-free survival and is regarded as the most malignant subtype of breast cancer. The purpose of this study is to explore prognostic biomarkers that can predict metastasis of triple-negative breast cancer. Methods. The human triple-negative breast cancerSUM149PT cells were used for the study. The cancer stem cell spheres (sum149-Stem) and paired adherent cancer cells (sum149-Tumor) were collected to extract total RNAs. RNA-seq was used to analysis the mRNA expression of cancer stem cells and paired adherent cancer cells. Two different gene expression omnibus datasets (https://www.ncbi.nlm.nih.gov/gds), GSE58812 and GSE33926, were used to explore the mechanism of different expression genes between stem cells and adherent cancer cells. Seven genes showed prognostic function in all datasets. The STITCH database (https://www.stitchdata.com/) was used to explore the possible metastasis-inhibiting drugs that can target the seven genes. Each gene expression was compared by Pearson analysis. The receiver operating characteristic curve (ROC) and Kaplan–Meier survival curve were performed to assess the metastasis prognostic ability of the seven-gene modeling two different GEO datasets. Results. A subset of 7 stemness-related genes (SRGs) containing UCN, ST3GAL5, FDPS, HK2, MALL, LMTK3, and CRHR2 were identified in three independent cohorts. Univariate Cox analysis showed that ST3GAL5 plays an antitumor role in TNBC metastasis, and the other 6 genes promote the metastatic progression of TNBC. The ability of the 7-SRGs gene Cox model to predict TNBC metastasis was constructed with the GSE58812 dataset. Most of the genes showed significant expression in patients with different risk levels. Additionally, the model showed predictive value in another GEO dataset of TNBC patients. ROC curves indicated that the seven-gene model has a significant predictive value of TNBC metastasis. Conclusions. Expression analysis of the 7-SRGs signature model at diagnosis has predictive value for metastasis in TNBC patients.

SEC61 translocon subunit gamma enhances low-dose cisplatin-induced cancer-stem cell properties of head and neck squamous cell carcinoma via enhancing Ca2+-mediated autophagy

Background/purposeHigh SEC61 translocon subunit gamma (SEC61G) expression is associated with an unfavorable prognosis in patients with head and neck squamous cell carcinoma (HNSCC), but the underlying mechanisms remain poorly understood. Materials and methodsHNSCC representative cell lines SCC15 and CAL27 were used to explore the regulation of SEC61G on Ca2+ leak from the endoplasmic reticulum (ER). Ca2+−activated autophagy was monitored by fluorescent labeling of autophagosomes and western blotting assays. CSC marker expression, sphere formation, colony formation, and transwell of invasion were detected to investigate the role of SEC61G in regulating cancer-stem cell (CSC) properties. ResultsAmong the SEC61 complex genes, only SEC61G upregulation is consistently associated with unfavorable progression-free interval and disease-specific survival in patients with HNSCC. Low-dose cisplatin (CDDP) treatment induced SEC61G upregulation in SCC15 and CAL27 cells. SEC61G knockdown significantly impaired CDDP-induced Ca2+ from the ER and the phosphorylation of ERK1/2 and AMPK. CDDP-induced autophagy in HNSCC cells were hampered by SEC61G shRNA, in terms of impaired autophagosome formation, lowered LC3-II/GAPDH ratio and restored p62 expression. CDDP-induced CSC properties, including CSC marker expression, sphere formation, colony formation, and invasive capabilities could be suppressed by shSEC61G and chloroquine, a specific autophagy inhibitor. ConclusionFindings of this study revealed the contribution of SEC61G in promoting cisplatin-induced CSC properties of HNSCC cells via promoting Ca2+-mediated autophagy.

A curcumin analogue GO-Y030 depletes cancer stem cells by inhibiting the interaction between the HSP70/HSP40 complex and its substrates.

Cancer stem cells (CSCs) are proposed to be involved in tumor initiation and play important roles in cancer relapse, metastasis, and drug resistance. Therefore, the targeting of CSCs has potential for effective anticancer therapies. Curcumin is one of the most widely characterized phytochemicals with tumor-suppressive potential. GO-Y030 is a novel curcumin analogue exhibiting a much stronger growth-inhibitory effect than curcumin. In the present study, we verified the potency of GO-Y030 against a CSC population. We observed that GO-Y030 suppressed CSC sphere-forming ability in several cancer cell lines. Interestingly, a specific inhibitor of heat shock protein (HSP) 70 also exhibited effects similar to GO-Y030 (i.e. inhibition of CSC sphere formation and upregulation of HSP70 and HSP40 protein expression), suggesting that HSP70 and/or HSP40 might be target molecules of GO-Y030. We then performed an in vitro HSP70/HSP40-mediated refolding activity assay and observed that chaperone activity was efficiently inhibited by GO-Y030. Finally, we performed a substrate-binding assay to show that GO-Y030 reduced the binding of both HSP70 and HSP40 with their substrates. HSPs prevent denaturation or unfolding of client proteins under stressful conditions such as high temperature. Because CSCs by nature adapt to various stresses by reinforcing protein-folding activity, the function of HSP70/HSP40 is important for the maintenance of CSC population. Our data suggest that GO-Y030 may impair stress tolerance in CSCs by inhibiting the interaction of HSP70/HSP40 with their substrate proteins and disrupting the function of HSP70/HSP40, thereby contributing to a reduction of the CSC population.

Identification of the polyether ionophore lenoremycin through a new screening strategy for targeting cancer stem cells.

Targeting and eradicating cancer stem cells (CSCs), also termed tumor-initiating cells, are promising strategies for preventing cancer progression and recurrence. To identify candidate compounds targeting CSCs, we established a new screening strategy with colorectal CSC spheres and non-CSC spheres in three-dimensional (3D) culture system. Through chemical screening using our system with in-house microbial metabolite library, we identified polyether cation ionophores that selectively inhibited CSC sphere formation, whereas CSC spheres were resistant to conventional anticancer agents. One of the hit compounds, the most selective and effective microbial metabolite lenoremycin, decreased CSC populations via inducing reactive oxygen species production. This study demonstrated that our newly established screening system is useful for discovering agents that selectively eliminate CSCs.

Abstract 3171: ARL2 is required for homologous recombination repair and colon cancer stem cell maintenance

Abstract ARL2 is a small GTP binding protein mediates many cytosolic functions including localization of Ras, dynamics of microtubule and fission of mitochondria but its nuclear function is not well elucidated yet. Here we present the novel role of ARL2 in human cancer nucleus and cancer stem cells (CSC). The cancer tissue gene expression data in The Human Protein Atlas and The Cancer Genome Atlas showed possible pro-cancer and nuclear role of ARL2 in colon cancers. The single cell RNA seq analysis suggests its function in colon stem cells. The activation mutant of K-Ras harboring colon cancer cells expressed less ARL2 than the wild type cells but increased ARL2 expression in CSC spheres. The deprivation of ARL2 in colon cancer cell lines using RNAi preferentially eliminated the CSC spheres while it resulted in minimal impact on bulk cultured cell (BCC) numbers. Suppression of ARL2 halted the BCC at M phase as expected but did not change cell cycle in CSC spheres. However, it led to accumulation of a DNA double strand break marker and apoptosis only in CSC spheres which suggest that ARL2’s requirement for double strand DNA break repair in CSC. Out of 6 somatic RAD51 family genes which all mediate the critical early events of homologous recombination repair (HRR), 5 showed significant and positive association with ARL2 in gene expression in human colon cancer tissue suggesting functional link between ARL2 and HRR. Indeed, ARL2 was required for HRR when double strand DNA breaks were induced. These data collectively showed that ARL2 may contribute to the HRR in the nucleus and the ARL2 mediated HRR is more critical for colon CSC survival. Citation Format: Seok Gyeong Choi, Hani Lee, Sojung Ha, Sukjoon Yoon, Woo-Young Kim. ARL2 is required for homologous recombination repair and colon cancer stem cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3171.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Cancer-Associated Fibroblasts Facilitate Squamous Cell Carcinoma Lung Metastasis in Mice by Providing TGFβ-Mediated Cancer Stem Cell Niche.

Cancer-associated fibroblasts (CAFs) have been shown to enhance squamous cell carcinoma (SCC) growth, but it is unclear whether they promote SCC lung metastasis. We generated CAFs from K15.KrasG12D.Smad4–/– mouse SCCs. RNA expression analyses demonstrated that CAFs had enriched transforming growth factor-beta (TGFβ) signaling compared to normal tissue-associated fibroblasts (NAFs), therefore we assessed how TGFβ-enriched CAFs impact SCC metastasis. We co-injected SCC cells with CAFs to the skin, tail vein, or the lung to mimic sequential steps of lung metastasis. CAFs increased SCC volume only in lung co-transplantations, characterized with increased proliferation and angiogenesis and decreased apoptosis compared to NAF co-transplanted SCCs. These CAF effects were attenuated by a clinically relevant TGFβ receptor inhibitor, suggesting that CAFs facilitated TGFβ-dependent SCC cell seeding and survival in the lung. CAFs also increased tumor volume when co-transplanted to the lung with limiting numbers of SCC cancer stem cells (CSCs). In vitro, CSC sphere formation and invasion were increased either with co-cultured CAFs or with CAF conditioned media (which contains the highest TGFβ1 concentration) and these CAF effects were blocked by TGFβ inhibition. Further, TGFβ activation was higher in primary human oral SCCs with lung metastasis than SCCs without lung metastasis. Similarly, TGFβ activation was detected in the lungs of mice with micrometastasis. Our data suggest that TGFβ-enriched CAFs play a causal role in CSC seeding and expansion in the lung during SCC metastasis, providing a prognostic marker and therapeutic target for SCC lung metastasis.

Ascorbic Acid Inhibits Liver Cancer Growth and Metastasis in vitro and in vivo, Independent of Stemness Gene Regulation.

Experimental and clinical evidence has indicated that the natural product ascorbic acid (AA) is effective in preventing and treating various types of cancers. However, the effect of AA on liver cancer metastasis has not yet been reported. Cancer stem cells (CSCs) play pivotal roles in cancer metastasis. Here, we demonstrated that AA selectively inhibited the viability of both liver cancer cells and CSCs, reduced the formation of cancer cell colonies and CSC spheres, and inhibited tumor growth in vivo. Additionally, AA prevented liver cancer metastasis in a xenotransplantation model without suppressing stemness gene expression in liver CSCs. Further study indicated that AA increased the concentration of H2O2 and induced apoptosis in liver CSCs. Catalase attenuated the inhibitory effects of AA on liver CSC viability. In conclusion, AA inhibited the viability of liver CSCs and the growth and metastasis of liver cancer cells in vitro and in vivo by increasing the production of H2O2 and inducing apoptosis. Our findings provide evidence that AA exerts its anti-liver cancer efficacy in vitro and in vivo, in a manner that is independent of stemness gene regulation.

Combined Poziotinib with Manidipine Treatment Suppresses Ovarian Cancer Stem-Cell Proliferation and Stemness.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women worldwide, with an overall 5 year survival rate below 30%. The low survival rate is associated with the persistence of cancer stem cells (CSCs) after chemotherapy. Therefore, CSC-targeting strategies are required for successful EOC treatment. Pan-human epidermal growth factor receptor 4 (HER4) and L-type calcium channels are highly expressed in ovarian CSCs, and treatment with the pan-HER inhibitor poziotinib or calcium channel blockers (CCBs) selectively inhibits the growth of ovarian CSCs via distinct molecular mechanisms. In this study, we tested the hypothesis that combination treatment with poziotinib and CCBs can synergistically inhibit the growth of ovarian CSCs. Combined treatment with poziotinib and manidipine (an L-type CCB) synergistically suppressed ovarian CSC sphere formation and viability compared with either drug alone. Moreover, combination treatment synergistically reduced the expression of stemness markers, including CD133, KLF4, and NANOG, and stemness-related signaling molecules, such as phospho-STAT5, phospho-AKT, phospho-ERK, and Wnt/β-catenin. Moreover, poziotinib with manidipine dramatically induced apoptosis in ovarian CSCs. Our results suggest that the combinatorial use of poziotinib with a CCB can effectively inhibit ovarian CSC survival and function.

Histone Demethylase KDM4C Is Required for Ovarian Cancer Stem Cell Maintenance.

Ovarian cancer is a highly deadly disease, which is often diagnosed at a late stage with metastases. However, most ovarian cancers relapse after surgery combined with platinum-based chemotherapy. Cancer stem cells (CSCs) are stem-like cells that possess high tumorigenic capability and display higher resistant capability against current therapies. However, our knowledge of ovarian CSCs and their molecular mechanism remains sparse. In the current study, we found that KDM4C, a histone demethylase, was required for ovarian cancer stem cell (CSC) maintenance. Depletion of KDM4C significantly reduced the CSC population and sphere formation in vitro. Moreover, we found that KDM4C can regulate the expression of stem cell factor OCT-4 via binding to its promoter. These data indicate that KDM4C is relevant for ovarian CSC maintenance and underscore its importance as a potential therapeutic target.

Abstract 6377: Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates

Abstract It has been established that a cancer tissue contains, in general, a small subpopulation of cancer stem cells (CSCs) having tumor initiation capacity. Because CSCs are resistant to chemoradiotherapy and play important roles in cancer relapse and metastasis, strategies targeting CSCs are crucial for effective anticancer therapies. Curcumin (diferuloylmethane) isolated from the rhizome of the plant turmeric (Curcuma longa) is one of the most widely characterized phytochemicals and well known to suppress cancer cell proliferation, invasion, angiogenesis, and metastasis by regulating multiple cell signaling pathways. However, its bioavailability is limited due to poor absorption. To improve this disadvantage, Ohori et al. (Mol Cancer Ther 2006;5:2563-71.) synthesized a new curcumin analog GO-Y030 (1,5-bis(3,5-bis(methoxymethoxy)phenyl)-1,4-pentadiene-3-one), which has an about 30-fold greater growth-inhibitory effect than curcumin. In the current study, we verified the potency of GO-Y030 to act against CSC population. We first discovered that GO-Y030 could dose-dependently suppress a sphere-forming ability in human HuH7 hepatocellular carcinoma and human PC3 prostatic carcinoma cell lines. Interestingly, the specific inhibitor of the stress-inducible heat shock protein (HSP) 70 (also called HSP72, HSP70-1, or HSPA1A) exhibited effects similar to GO-Y030, i.e., inhibition of CSC sphere formation and upregulation of HSP70 and HSP40 protein expression. As this result suggested that target molecules of GO-Y030 might be HSP70 and its co-chaperones such as HSP40, we next performed in vitro HSP70/HSP40-mediated refolding activity assay and revealed that GO-Y030 efficiently inhibited HSP70/HSP40 chaperone activity. Finally, performing the substrate-binding assay, we have proven that GO-Y030 reduces the substrate-recognition and binding activity of HSP70. HSPs are molecular chaperones that function to prevent denaturation or unfolding of client proteins when they are threatened by stress or high temperature. Since CSCs by nature adapt to oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress by enhancing synthesis of protein-folding chaperones, the function of HSP70/HSP40 is important for the maintenance of CSC population. Our data suggest that GO-Y030 should impair stress tolerance in CSCs by inhibiting the interaction of HSP70 with substrate proteins and disrupting the function of HSP70/HSP40 and thus should finally contribute to reduction of CSC population. Citation Format: Maya Suzuki, Yohei Yamamoto, Aki Nishijima, Hiroyuki Shibata, Yasufumi Omori. Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates [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 6377.

Abstract 6028: Interferon induces lineage plasticity of castration-resistant prostate cancer

Abstract Prostate cancer (PCa) remains to be the leading cause of cancer incidence in men of the United States. Up to date, androgen deprivation therapy (ADT) remained to be the primary strategy for advanced PCa. Despite drugs targeting androgen receptor (AR) are initially effective for PCa, the disease ultimately recurs to the end stage of castration-resistant PCa (CRPC) that often exhibits neuroendocrine phenotypes, suggesting lineage plasticity is involved in CRPC progression. Emergence of cancer stem cells (CSCs) is highly correlated with the lethal phenotypes; the involvement of PCSCs could explain the resistance of CRPC to chemotherapy or radiotherapy. Therefore, elucidating the mechanisms underlying the acquisition of lineage plasticity in prostate cancer (PCSC) is critical for establish the therapeutic strategy targeting the variant types of CRPC. Based on RNA-seq data from a panel of PCa cells with neuroendocrine phenotypes, interferon (IFN) pathway, such as signal transducer and activator of transcription 1 (STAT1), is highly elevated. Clinical observations have found significantly elevated serum IFN levels in PCa patients after ADT and/or irradiation. Meanwhile, IFN-related DNA damage resistance signature (IRDS) encompassed a subset of STAT1-driven genes with pro-survival functions are known to responsible for intrinsic resistance to chemotherapy and radiotherapy in many malignancies. Altogether, we determined to investigate the involvement of STAT1 signaling in the lineage plasticity of CRPC. We applied tumor sphere assay to study the self-renewal capacity of PCSCs in vitro and subcutaneous injection model to examine tumor incidence and tumor growth in vivo. We observed that IRDS and STAT1-driven genes are significantly upregulated in metastatic tumor specimens of PCa patients and CRPC lines acquired lineage plasticity. Inhibition of JAK-STAT1 by specific small molecule inhibitors such as Ruxolitinib or Fludarabine significantly suppresses the self-renewal capacity of CSC spheres in vitro, and attenuates tumor growth in vivo. In particular, we demonstrated that IFIT5, a bona fide IFN inducible gene, appears to be the key molecule in facilitating the acquisition of stemness properties in PCSCs via regulating Bmi1, Sox2 and Nanog through targeting the turnover of miR-128 and miR-101. In addition, loss of IFIT5 attenuates sphere forming ability in vitro, and decreases tumor incidence in vivo. Moreover, IFIT5, STAT1 and several IRDS genes are highly expressed in CRPC lines with neuroendocrine features when compared to adenocarcinoma lines, suggesting association of STAT1 activation with the lineage plasticity of CRPC. Overall, we demonstrated the impact of STAT1 signaling-induced IFIT5-mediated microRNA turnover on the acquisition of stemness properties of PCSCs. The outcome of this study may provide a novel therapeutic strategy for managing the therapy-resistant CRPC. Citation Format: U-Ging Lo, Yu-An Chen, Ping Mu, Ho Lin, Chih-Ho Lai, Jer-Tsong Hsieh. Interferon induces lineage plasticity of castration-resistant prostate cancer [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 6028.

Broccoli extract increases drug-mediated cytotoxicity towards cancer stem cells of head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinomas (HNSCC) are malignant neoplasms with poor prognosis. Treatment-resistant cancer stem cell (CSC) is one reason for treatment failure. Considerable attention has been focused on sulforaphane (SF), a phytochemical from broccoli possessing anticancer properties. We investigated whether SF could enhance the chemotherapeutic effects of cisplatin (CIS) and 5-fluorouracil (5-FU) against HNSCC–CSCs, and its mechanisms of action.MethodsCD44+/CD271+ FACS-isolated CSCs from SCC12 and SCC38 human cell lines were treated with SF alone or combined with CIS or 5-FU. Cell viability, colony- and sphere-forming ability, apoptosis, CSC-related gene and protein expression and in vivo tumour growth were assessed. Safety of SF was tested on non-cancerous stem cells and in vivo.ResultsSF reduced HNSCC–CSC viability in a time- and dose-dependent manner. Combining SF increased the cytotoxicity of CIS twofold and 5-FU tenfold, with no effects on non-cancerous stem cell viability and functions. SF-combined treatments inhibited CSC colony and sphere formation, and tumour progression in vivo. Potential mechanisms of action included the stimulation of caspase-dependent apoptotic pathway, inhibition of SHH pathway and decreased expression of SOX2 and OCT4.ConclusionsCombining SF allowed lower doses of CIS or 5-FU while enhancing these drug cytotoxicities against HNSCC–CSCs, with minimal effects on healthy cells.