Expression Of Cancer Stem Cell Markers Research Articles

Resistance to FOXM1 inhibitors in breast cancer is accompanied by impeding ferroptosis and apoptotic cell death.

Cancer treatments often become ineffective because of acquired drug resistance. To characterize changes in breast cancer cells accompanying development of resistance to inhibitors of the oncogenic transcription factor, FOXM1, we investigated the suppression of cell death pathways, especially ferroptosis, in FOXM1 inhibitor-resistant cells. We also explored whether ferroptosis activators can synergize with FOXM1 inhibitors and can overcome FOXM1 inhibitor resistance. In estrogen receptor-positive and triple-negative breast cancer cells treated with FOXM1 inhibitor NB73 and ferroptosis activators dihydroartemisinin and JKE1674, alone and in combination, we measured suppression of cell viability, motility, and colony formation, and monitored changes in gene and protein pathway expressions and mitochondrial integrity. Growth suppression of breast cancer cells by FOXM1 inhibitors is accompanied by increased cell death and alterations in mitochondrial morphology and metabolic activity. Low doses of FOXM1 inhibitor strongly synergize with ferroptosis inducers to reduce cell viability, migration, colony formation, and expression of proliferation-related genes, and increase intracellular Fe+2 and lipid peroxidation, markers of ferroptosis. Acquired resistance to FOXM1 inhibition is associated with increased expression of cancer stem-cell markers and proteins that repress ferroptosis, enabling cell survival and drug resistance. Notably, resistant cells are still sensitive to growth suppression by low doses of ferroptosis activators, effectively overcoming the acquired resistance. Delineating changes in viability and cell death pathways that can overcome drug resistance should be helpful in determining approaches that might best prevent or reverse resistance to therapeutic targeting of FOXM1 and ultimately improve patient clinical outcomes.

Neurotrophin-3 Facilitates Stemness Properties and Associates with Poor Survival in Lung Cancer.

Cancer stem cells (CSCs) shape the tumor microenvironment via neuroendocrine signaling and orchestrate drug resistance and metastasis. Cytokine antibody array demonstrated the upregulation of neurotrophin-3 (NT-3) in lung CSCs. This study aims to dissect the role of NT-3 in lung CSCs during tumor innervation. Western blotting, quantitative reverse transcription-PCR, and flow cytometry were used to determine the expression of the NT-3 axis in lung CSCs. NT-3-knockdown and NT-3-overexpressed cells were derived lung CSCs, followed by examining the stemness gene expression, tumorsphere formation, transwell migration and invasion, drug resistance, soft agar colony formation, and in vivo tumorigenicity. Human lung cancer tissue microarray and bioinformatic databases were used to investigate the clinical relevance of NT-3 in lung cancer. NT-3 and its receptor tropomyosin receptor kinase C (TrkC) were augmented in lung tumorspheres. NT-3 silencing (shNT-3) suppressed the migration and anchorage-independent growth of lung cancer cells. Further, shNT-3 abolished the sphere-forming capability, chemo-drug resistance, invasion, and in vivo tumorigenicity of lung tumorspheres with a decreased expression of CSC markers. Conversely, NT-3 overexpression promoted migration and anchorage-independent growth and fueled tumorsphere formation by upregulating the expression of CSC markers. Lung cancer tissue microarray analysis revealed that NT-3 increased in patients with advanced-stage, lymphatic metastasis and positively correlated with Sox2 expression. Bioinformatic databases confirmed a co-expression of NT-3/TrkC-axis and demonstrated that NT-3, NT-3/TrkC, NT-3/Sox2, and NT-3/CD133 worsen the survival of lung cancer patients. NT-3 conferred the stemness features in lung cancer during tumor innervation, which suggests that NT-3-targeting is feasible in eradicating lung CSCs.

Diallyl trisulfide induces pyroptosis and impairs lung CSC-like properties by activating the ROS/Caspase 1 signaling pathway

Lung cancer stem cells (CSCs) drive continuous cancer growth and metastatic dissemination; thus, there is an urgent requirement to acquire effective therapeutic strategies for targeting lung CSCs. Diallyl trisulfide (DATS), a garlic organosulfide, possesses suppressive potential in lung cancer; however, its underlying mechanism is still unclear. In this study, we identified DATS as a pyroptosis inducer in lung cancer cells. DATS-treated A549 and H460 cells exhibited pyroptotic cell death, with characteristic large bubbles appearing on their plasma membrane and LDH release. DATS induced cell death, arrested the cell cycle at the G2/M phase, and inhibited colony formation in lung cancer cells. Meanwhile, we found that DATS significantly suppressed the malignant features by impairing lung CSC-like properties, including sphere formation ability, CD133 positive cell number, and lung CSCs marker expression. Mechanistically, DATS induced cell pyroptosis via increasing the expression of NLRP3, ASC, Pro Caspase 1, Cleaved Caspase 1, GSDMD, GSDMD-N, and IL-1β. The verification experiments showed that the effects of DATS on pyroptosis and lung CSC-like properties were weakened after Caspase 1 inhibitor VX-765 treatment, indicating that DATS activated NLRP3 inflammasome-mediated pyroptosis by targeting Caspase 1 in lung cancer cells. Moreover, DATS increased ROS overproduction and mitochondrial dysfunction, which contributed to DATS-induced pyroptosis of lung cancer cells. NAC treatment reversed the effects of DATS on pyroptosis and CSC-like properties. In vivo experiment further confirmed that DATS restrained tumor growth. Together, our results suggest that DATS promotes pyroptosis and impairs lung CSC-like properties by activating ROS/Caspase 1 signaling pathway, thereby retarding lung cancer progression.

Adenovirus-mediated expression of MOAP-1, Bax and RASSF1A antagonizes chemo-drug resistance of human breast cancer cells expressing cancer stem cell markers

Cancer is one of the major leading causes of mortality globally and chemo-drug-resistant cancers pose significant challenges to cancer treatment by reducing patient survival rates and increasing treatment costs. Although the mechanisms of chemoresistance vary among different types of cancer, cancer cells are known to share several hallmarks, such as their resistance to apoptosis as well as the ability of cancer stem cells to produce metastatic daughter cells that are resistant to chemotherapy. To address the issue of chemo-drug resistance in cancer cells, a tetracistronic expression construct, Ad-MBR-GFP, encoding adenovirus-mediated expression of MOAP-1, Bax, RASSSF1A, and GFP, was generated to investigate its potential activity in reducing or inhibiting the chemo-drug resistant activity of the human breast cancer cells, MCF-7-CR and MDA-MB-231. When infected by Ad-MBR-GFP, the cancer cells exhibited round cell morphology and nuclei condensation with positive staining for annexin-V. Furthermore, our results showed that both MCF-7-CR and MDA-MB-231 cells stained positively for CD 44 and negatively for CD 24 (CD44+/CD24-) with high levels of endogenous ALDH activity whereas SNU-1581 breast cancer cells were identified as CD 44-/CD 24- cells with relatively low levels of endogenous ALDH activity and high sensitivity toward chemo-drugs, suggesting that both CD 44 and ALDH activity contribute to chemo-drug resistance. Moreover, both MCF-7-CR and MDA-MB-231 cells showed strong chemo-drug sensitivity to cisplatin when the cells were infected by Ad-MBR-GFP, leading to 9-fold and 2-fold reduction in the IC 50 values when compared to cisplatin treatment alone, respectively. The data were further supported by 3D (soft agar) and spheroid cell models of MCF-7-CR and MDA-MB-231 cells which showed a 2-fold reduction of a number of cell colonies and spheroid size when treated with both Ad-MBR-GFP and cisplatin, and compared to control. Other than chemo-sensitivity, Ad-MBR-GFP-infected cancer cells retarded cell migration. Flow cytometry analysis showed that the mechanism of action of Ad-MBR-GFP involved cell cycle arrest at the G1 phase and inhibition of cellular DNA synthesis. Taken together, our investigation showed that Ad-MBR-GFP mediated chemo-drug sensitization in the infected cancer cells involved the activation of apoptosis signaling, cell cycle arrest, and inhibition of DNA synthesis, suggesting that Ad-MBR-GFP is potentially efficacious for the treatment of chemo-drug resistant cancers.

Abstract 5431: Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a common type of cancer globally and is associated with a poor prognosis due to its high recurrence rate and resistance to treatment. Solid evidence showed the existence of cancer stem cells (CSCs) that are crucial for therapeutic resistance. However, there are only a few kinase inhibitors that have been found to specifically target liver T-ICs for the HCC treatment. To understand the regulatory mechanism of liver CSCs, we employed RNA-Seq analysis to compare the gene expression profiles, with a particular focus on protein kinases, between (1) sorted CD133+ liver CSCs and CD133- differentiated cells extracted from two HCC cell lines (Huh7 and PLC8024), as well as cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. Our analysis identified Serine/Threonine kinase 39 (STK39) as a commonly up-regulated protein kinase in both CD133+ liver CSCs and CSC-enriched hepatospheres. Based on our analysis of the TCGA data, a positive correlation has consistently been observed between STK39 mRNA expression and the expression of liver CSC markers, including CD24, CD133, and CD47. Utilizing publicly available transcriptome datasets from the NCBI Gene Expression Omnibus (GEO), including GSE14520 and GSE25097, we found that overexpression of STK39 is detected in HCC tumor tissues (p<0.001) and is significantly associated with poorer overall survival and disease-free survival. Through the use of lentiviral based overexpression and knockdown approaches, we have demonstrated the role of STK39 in regulating liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness, and the expression of liver CSC markers. STK39 expression was found to be significantly elevated in in HCC cells that were resistant to both sorafenib and lenvatinib, and its inhibition resulted in increased sensitivity of HCC cells to these treatments. To identify the downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. The analysis led to the identification of PARP1 as a novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, it was observed that the phosphorylation of PARP1 at Threonine 368 (T368) was significantly inhibited, indicating its role as a downstream effector of STK39-mediated liver CSC functions. Interestingly, STK39-mediated PARP1 phosphorylation led to development of resistance to PARP1 inhibitor, rucaparib. Additionally, STK39 plays a critical role in regulating PARP1-mediated chromatin decondensation, which enables it to open chromatin structure at the promoter regions of SOX2/OCT4. In summary, treatment of molecular targeted drugs including rucaparib and lenvatinib with STK39 inhibitor may be a potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma [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 5431.

Abstract 2520: CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast cancer

Abstract Introduction: Cancer stem cells (CSC) constitute only 0.1-1% of all the cells in a bulk tumor, yet they play a crucial role in treatment resistance and relapses. Its role in breast cancer (BC) has been studied but a consensus remains elusive. We focused on gene expression of a CSC marker CD133 and studied its clinical significance in ER-positive/HER2-negative (ER+/HER2-) BC, the most prevalent subtype. We utilized multiple independent large patient cohorts of gene expression and clinical variables for transcriptome analysis. Methods: A total of 2969 patients from The Cancer Genome Atlas (TCGA, n=1065) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n=1904) BC cohorts, as well as BC patients who underwent neoadjuvant chemotherapy (NAC; GSE25066, GSE20194, GSE32646 cohorts) were analyzed. Results: By analyzing SCP1039 and SCP1106 single-cell sequencing cohorts, we found that epithelial cells were the predominant source of CD133 gene expression in the tumor microenvironment. High expression of CD133 in ER+/HER2- BC correlated with elevated gene expressions of the other CSC markers; CD24, NOTCH1, DLL1, and ALDH1A1 (all p<0.001), and significantly enriched WNT/β-Catenin, Hedgehog, and Notch signaling (all FDR<0.25), which are all known pathways related with CSC in TCGA and validated in METABRIC cohort. Consistent with CSC phenotype, CD133-high BC was negatively enriched with Hallmark cell proliferation-related gene sets, such as E2F Targets, G2M Checkpoint, MYC Targets V1, Ki67 gene expression and proliferation score (FDR<0.25 and p<0.001, respectively) in both cohorts. CD133-high BC was negatively associated with DNA repair genes, such as BRCA1, E2F7 in both cohorts (all p<0.001). In agreement, CD133-high BC was associated with less homologous recombination deficiency, lower silent and non-silent mutation rates. Surprisingly, CD133-high BC exhibited a proinflammatory microenvironment, as represented by higher cytolytic activity in the METABRIC and higher lymphocyte infiltrations, TGF-beta response, and TCR richness in TCGA (both p<0.001), but less Th1 cells, M1 macrophages and dendritic cell infiltrations consistently in both cohorts. It also enriched signaling pathways related to inflammation and immune responses such as inflammatory response, allograft rejection, TNF-alpha, IL-6, TGF-beta signaling, and complement signaling (all FDR<0.25). CD133-high tumors had better pathological complete response after NAC in GSE25066 cohort and improved Disease-Free Survival and Overall Survival in both TCGA and METABRIC cohorts (all p<0.05). Conclusion: CD133-high ER+/HER2- BC is associated with CSC phenotype, reduced cell proliferation, and impaired DNA repair, but enhanced inflammation, potentially explaining the better response to NAC and favorable patient prognosis. Citation Format: Takumi Sato, Masanori Oshi, Jing Lee, Arya M. Roy, Kohei Chida, Itaru Endo, Masato Obayashi, Kazuaki Takabe. CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast cancer [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 2520.

Abstract 5877: Disulfiram and its metal complexes reversed hypoxia induced stemness and synergized with first line drugs in primary patient derived pancreatic ductal adenocarcinoma cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate (12%) and is predicted to become the second leading cause of cancer related deaths by 2030. The main treatment option for advanced PDAC, gemcitabine (GEM) and nab-paclitaxel (PAC) chemotherapy, has limited efficacy due to adverse toxicities and chemoresistance. Extensive desmoplasia in the PDAC microenvironment induces hypoxia that drives epithelial to mesenchymal transition (EMT) and promotes cancer stem cells (CSCs) leading to metastasis and acquired resistance. Drugs that target CSCs and EMT to overcome chemoresistance could improve patient outcome. Disulfiram (DSF), an FDA approved anti alcoholism drug, has excellent anticancer activity on chelation with Cu2+/Zn2+ ions to form the anticancer metabolites, Copper or Zinc Diethyldithiocarbamate (CuDDC or ZnDDC). The anticancer application of the current oral version of DSF is prevented by its short plasma half-life (< 4 minutes) and separate administration of oral Cu/Zn supplement leading to bioavailability issues. A stable, long circulating, injectable formulation of DSF, CuDDC or ZnDDC may overcome these pharmacokinetic issues. In this study albumin was used to develop soluble, stable and homogenous CuDDC and ZnDDC nanoparticles (Alb-CuDDC, Alb-ZnDDC) and their efficacy was compared with free DSF-Cu in a panel of normoxic, hypoxic and spheroid cultures of patient PDAC cells derived from primary, circulating and metastatic sites. Hypoxia and spheroid cultures of PDAC cells were highly resistant to GEM and PAC and had increased expression of CSC markers such as ALDH, CD133, ABCG2 and CA9. Alb-CuDDC and Alb-ZnDDC were cytotoxic to all PDAC cells (IC50s ~300 nM and ~7000 nM, respectively) in normoxia and hypoxia and synergized with GEM and PAC. While DSF-Cu and Alb-CuDDC significantly reduced the expression of CSC markers and inhibited sphere reformation and migration of PDAC cells, Alb-ZnDDC was less effective in inhibiting these features. Therefore, the ability of PDAC cells to acquire resistance to Alb-ZnDDC was investigated. After long term exposure (> 4 months) to increasing doses of each drug (DSF-Cu, Alb-CuDDC and Alb-ZnDDC), PANC1 and BXPC3 PDAC cells acquired resistance only to Alb-ZnDDC. PANC1ZR6 and BXPC3ZR5 resistant cells showed increased expression of CSC markers and significant cross-resistance to PAC and GEM suggesting multidrug resistance. Importantly, no cross-resistance to Alb-CuDDC or DSF-Cu was observed, suggesting alternative mechanisms for Alb-ZnDDC. RNA sequencing is underway to identify potential mechanisms involved. To conclude, albumin formulation improved the solubility and stability of DSF-metal complexes. Alb-CuDDC targets CSCs and overcomes resistance in PDAC, similar to free DSF-Cu. Alb-ZnDDC may have alternative mechanisms which are yet to be elucidated. Citation Format: Benjamin J. Small, Ogechi Nkeonye, Vinodh Kannappan, Shalini Kumarasamy, Kate Butcher, Karim Azar, Gowtham Rajendran, Yoshitha Lakshmanan Lakshmanan, Mark Morris, Christopher Heeschen, Yaohe Wang, Weiguang Wang. Disulfiram and its metal complexes reversed hypoxia induced stemness and synergized with first line drugs in primary patient derived pancreatic ductal adenocarcinoma cells [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 5877.

Abstract 4250: Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [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 4250.

Abstract 5439: Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers

Abstract Background: Elevated expression of cancer stem cell (CSC) markers is linked with progression, poor survival, and treatment resistance in glioblastoma (GBM). Our prior work identified KAI1 COOH-terminal interacting tetraspanin (KITENIN) as key to GBM progression. We investigated its influence on temozolomide (TMZ) resistance in GBM through modulation of CSC markers. Methods: We examined KITENIN and CSC markers in TMZ-resistant GBM cells and patient-derived GBM cells, and assessed therapeutic responses to TMZ in mice implanted with KITENIN-modulated GBM cells. The correlation of these markers with clinical outcomes was analyzed using our cohort and The Cancer Genome Atlas (TCGA) data. Results: High KITENIN expression correlated positively with elevated CSC marker levels in human GBM samples and KITENIN-modulated GBM cells. Inhibition of KITENIN using usnic acid reduced tumor progression by decreasing CSC marker expression. Our data and TCGA analyses linked co-directional increase of these factors with tumor recurrence and shorter survival in GBM patients with unmethylated MGMT. This increase was also observed in TMZ-resistant cell lines with unmethylated MGMT. Implantation of KITENIN-overexpressing cells led to TMZ resistance in our mouse model, associated with co-directionally increased CD44 and ALDH1A1 expression. The link between KITENIN and CD44/ALDH1A1 could be inhibited by DKC2511, a novel KITENIN inhibitor, and T5244, a c-Fos/AP-1 inhibitor. Conclusion: KITENIN might contribute to TMZ resistance in GBM patients with unmethylated MGMT, potentially through AP-1 activation and subsequent upregulation of CSC markers. Hence, targeting KITENIN could provide a novel strategy to overcome TMZ resistance in GBM patients with unmethylated MGMT. Citation Format: Eun-Jeong Ahn, Yeong Jin Kim, Tae-Young Jung, Jae-Hyuk Lee, Joon Haeng Rhee, Kyung-Keun Kim, Sung Sun Kim, Nah Ihm Kim, Hangun Kim, Kyung-Sub Moon, Kyung-Hwa Lee. Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers [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 5439.

Butyrate attenuates the stemness of lung cancer cells through lysosome Fe2+- and SLC7A11-mediated ferroptosis

Cancer stem cells (CSCs) are considered key contributors to tumor progression, and ferroptosis has been identified as a potential target for CSCs. We have previously shown that butyrate enhances the ferroptosis induced by erastin in lung cancer cell, this study aimed to investigate the impact of butyrate on the progression of lung CSCs. To investigate these effects, we constructed a series of in vitro experiments, including 3D non-adherent sphere-formation, cytometry analysis, assessment of CSC marker expression, cell migration assay, and in vivo tumorigenesis analyses. Additionally, the influence of butyrate on chemotherapeutic sensitivity were determined through both in vitro and in vivo experiments. Mechanistically, immunofluorescence analysis was employed to examine the localization of biotin-conjugated butyrate. We identified that butyrate predominantly localized in the lysosome and concurrently recruited Fe2+ in lysosome. Moreover, butyrate reduced the stability of SLC7A11 protein stability in lung cancer cells through ubiquitination and proteasome degradation. Importantly, the effects of butyrate on lung CSCs were found to be dependent on lysosome Fe2+- and SLC7A11-mediated ferroptosis. In summary, our results demonstrate that butyrate could induce the ferroptosis in lung CSCs by recruiting Fe2+ in lysosome and promoting the ubiquitination-lysosome degradation of SLC7A11 protein.

SOX2 and OCT4 mediate radiation and drug resistance in pancreatic tumor organoids

Pancreatic cancer has a five-year survival rate of only 10%, mostly due to late diagnosis and limited treatment options. In patients with unresectable disease, either FOLFIRINOX, a combination of 5-fluorouracil (5-FU), oxaliplatin and irinotecan, or gemcitabine plus nab-paclitaxel combined with radiation are frontline standard regimens. However, chemo-radiation therapy has shown limited success because patients develop resistance to chemotherapy and/or radiation. In this study, we evaluated the role of pancreatic cancer stem cells (CSC) using OCT4 and SOX2, CSC markers in mouse pancreatic tumor organoids. We treated pancreatic tumor organoids with 4 or 8 Gy of radiation, 10 μM of 5-FU (5-Fluorouracil), and 100 μM 3-Bromopyruvate (3BP), a promising anti-cancer drug, as a single treatment modalities, and in combination with RT. Our results showed significant upregulation of, OCT4, and SOX2 expression in pancreatic tumor organoids treated with 4 and 8 Gy of radiation, and downregulation following 5-FU treatment. The expression of CSC markers with increasing treatment dose exhibited elevated upregulation levels to radiation and downregulation to 5-FU chemotherapy drug. Conversely, when tumor organoids were treated with a combination of 5-FU and radiation, there was a significant inhibition in SOX2 and OCT4 expression, indicating CSC self-renewal inhibition. Noticeably, we also observed that human pancreatic tumor tissues exhibited heterogeneous and aberrant OCT4 and SOX2 expression as compared to normal pancreas, indicating their potential role in pancreatic cancer growth and therapy resistance. In addition, the combination of 5-FU and radiation treatment exhibited significant inhibition of the β-catenin pathway in pancreatic tumor organoids, resulting in sensitization to treatment and organoid death. In conclusion, our study emphasizes the crucial role of CSCs in therapeutic resistance in PC treatment. We recommend using tumor organoids as a model system to explore the impact of CSCs in PC and identify new therapeutic targets.

Abstract B035: Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer

Abstract Cancer stem cells (CSCs) are a subpopulation of cancer cells that are responsible for disease recurrence and chemoresistance. The mechanisms by which CSCs arise in endometrial cancer are not fully understood and elucidating key molecular events may provide insight into therapeutic targets. CD73, a cell surface 5’nucleotidase that generates adenosine, is frequently downregulated in endometrial tumorigenesis, and CD73 loss is associated with aggressive disease and poor patient outcomes. In developing and characterizing CRISPR/Cas-9 models for CD73, we discovered CD73 deletion (CD73−/−) in HEC-1-A cells causes growth arrest and prolonged survival which are features suggestive of a stemness phenotype. Thus, we hypothesized that loss of CD73 is critical for promoting CSCs in endometrial cancer. To evaluate CSC characteristics, we performed MTT and spheroid assays and interrogated CSC marker protein expression (e.g., CD44 and NOTCH3). CD73−/− HEC-1-A cells formed larger spheroids and expressed high levels of CD44 and NOTCH3 compared to wild-type (CD73+/+) cells. To assess whether loss of CD73 enzymatic activity is responsible for the enrichment of CSCs, CD73+/+ HEC-1-B and HEC-50 cells were treated with AoPCP, a catalytic inhibitor of CD73. Similar to genetic deletion of CD73 in HEC-1-A cells, AoPCP treatment resulted in HEC-1-B and HEC-50 cells forming larger spheroids and expressing high CD44 and NOTCH3. These data indicate that loss of CD73 enzyme activity is important to the enrichment of CSCs. In contrast, HEYA8 ovarian cancer cells showed the opposite effects, wherein treatment with AoPCP decreased spheroid size. These differences support the emerging paradigm that CD73 has tissue- and cancer-specific roles. As CD73 is a major node in purine metabolic signaling, we further hypothesized that loss of CD73 activity may be promoting CSC enrichment via changes to metabolic pathways. Mass spectrometry metabolomics revealed significantly different profiles of CD73+/+ and CD73−/− HEC-1-A cells. Conditioned media experiments showed CD73−/−-conditioned media induces growth arrest in HEC-1-A wild-type cells, whereas the growth arrest and CSC marker expression enrichment of CD73−/− cells was abrogated by CD73+/+-conditioned media or daily media replenishment. Metabolomic profiling of CD73+/+ and CD73−/− HEC-1-A cells with/without daily media replenishment revealed metabolic pathways, such as NAD+ biosynthesis, lipid metabolism, and sugar alcohol synthesis, are up-regulated by CD73 deficiency and parallels the enrichment of CSCs. Current studies are dissecting these metabolic dependencies in supporting endometrial cancer cell stemness. Altogether, these studies provide novel insight into how CSCs arise in endometrial cancer and why CD73 loss is detrimental in this disease. Citation Format: Emily M. Rabjohns, Blake R. Rushing, Sayali Joseph, Cyrus Vaziri, Jessica L. Bowser. Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B035.

Effect of Pioglitazone and Cetuximab on Colon Cancer Stem-like Cell (CCSLCs) Properties.

One of the main reasons for cancer resistance to chemotherapy is the presence of cancer stem cells (CSCs) in cancer tissues. It is also believed that CSCs are the unique originators of all tumor cells. On the other hand, the Epithelial-Mesenchymal Transition pathway (EMT) can act as the main agent of metastasis. Therefore, it is possible that targeting CSCs as well as the EMT pathway could help in cancer therapy. Considering that CSCs constitute only a small percentage of the total tumor mass, enrichment before study is necessary. In our previous study, CSCs were enriched in the human colon cancer cell line HT29 by induction of EMT. These CSC-enriched HT29 cells with mesenchymal morphology were named "HT29-shE". In the present study, these cells were used to investigate the effect of pioglitazone (Pio) and Cetuximab (Cet) in order to find CSC and EMT targeting agents. The viability and IC50 rate of cells treated with different concentrations of Pio and Cet were evaluated using the MTT test. EMT and CSC markers and cell morphology were assessed in Pio and Cet treated and untreated HT29-shE cells using flow cytometry, realtime PCR, immunocytochemistry, and microscopic monitoring. The findings showed that Pio and Cet at concentrations of 250 μM and 40 μg/ml, respectively, decrease cell viability by 50%. Also, they were able to reduce the expression of CSC markers (CD133 and CD44) in the CSC enriched HT29 cell line. Furthermore, Pio and Cet could efficiently reduce the expression of vimentin as a mesenchymal marker and significantly upregulate the expression of E-cadherin as an epidermal marker of EMT and its reverse mesenchymal-- to-epithelial transition (MET). In addition, the mesenchymal morphology of HT29-shE changed into epithelial morphology after Cet treatment. Pio and Cet could inhibit EMT and reduce CSC markers in the EMT induced/CSC enriched cell line. We expect that focus on finding EMT/CSC-targeting agents like these drugs can be helpful for cancer treatment.

Fbxw7 suppresses carcinogenesis and stemness in triple-negative breast cancer through CHD4 degradation and Wnt/β-catenin pathway inhibition

BackgroundCancer stem cells (CSCs) are a small population of cells in tumor tissues that can drive tumor initiation and promote tumor progression. A small number of previous studies indirectly mentioned the role of F-box and WD repeat domain-containing 7 (FBXW7) as a tumor suppressor in Triple-negative breast cancer (TNBC). However, few studies have focused on the function of FBXW7 in cancer stemness in TNBC and the related mechanism.MethodsWe detected FBXW7 by immunohistochemistry (IHC) in 80 TNBC patients. FBXW7 knockdown and overexpression in MD-MBA-231 and HCC1937 cell models were constructed. The effect of FBXW7 on malignant phenotype and stemness was assessed by colony assays, flow cytometry, transwell assays, western blot, and sphere formation assays. Immunoprecipitation-Mass Spectrometry (IP-MS) and ubiquitination experiments were used to find and verify potential downstream substrate proteins of FBXW7. Animal experiments were constructed to examine the effect of FBXW7 on tumorigenic potential and cancer stemness of TNBC cells in vivo.ResultsThe results showed that FBXW7 was expressed at low levels in TNBC tissues and positively correlated with prognosis of TNBC patients. In vitro, FBXW7 significantly inhibited colony formation, cell cycle progression, cell migration, EMT process, cancer stemness and promotes apoptosis. Further experiments confirmed that chromodomain-helicase-DNA-binding protein 4 (CHD4) is a novel downstream target of FBXW7 and is downregulated by FBXW7 via proteasomal degradation. Moreover, CHD4 could promote the nuclear translocation of β-catenin and reverse the inhibitory effect of FBXW7 on β-catenin, and ultimately activate the Wnt/β-catenin pathway. Rescue experiments confirmed that the FBXW7-CHD4-Wnt/β-catenin axis was involved in regulating the maintenance of CSC in TNBC cells. In animal experiments, FBXW7 reduced CSC marker expression and suppressed TNBC cell tumorigenesis in vivo.ConclusionsTaken together, these results highlight that FBXW7 degrades CHD4 protein through ubiquitination, thereby blocking the activation of the Wnt/β-catenin pathway to inhibit the stemness of TNBC cells. Thus, targeting FBXW7 may be a promising strategy for therapeutic intervention against TNBC.

Inhibition of MEK Signaling Attenuates Cancer Stem Cell Activity in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.

Evaluation of BMP-2 as a differentiating and radiosensitizing agent for colorectal cancer stem cells.

Despite effective clinical responses, a large proportion of patients undergo resistance to radiotherapy. The low response rate to current treatments in different stages of colorectal cancer depends on the prominent role of stem cells in cancer. In the present study, the role of BMP-2 as an ionizing radiation-sensitive factor in colorectal cancer cells was investigated. A sphere formation assay was used for the enrichment of HCT-116 cancer stem cells (CSCs). The effects of combination therapy (BMP-2+ radiation) on DNA damage response (DDR), proliferation, and apoptosis were evaluated in HCT-116 and CSCs. Gene expressions of CSCs and epithelial-mesenchymal transition (EMT) markers were also evaluated. We found that the sphere formation assay showed a significant increase in the percentage of CSCs. Moreover, expression of CSCs markers, EMT-related genes, and DNA repair proteins significantly decreased in HCT-116 cells compared to the CSCs group after radiation. In addition, BMP-2 promoted the radiosensitivity of HCT-116 cells by decreasing the survival rate of the treated cells at 2, 4, and 6 Gy compared to the control group in HCT-116 cells. Our findings indicated that BMP-2 could affect numerous signaling pathways involved in radioresistance. Therefore, BMP-2 ‎ can be considered an appealing therapeutic target for the treatment of radioresistant human colorectal cancer.

ZNF217-activated Notch signaling mediates sulforaphane-suppressed stem cell properties in colorectal cancer

Cancer stem cells (CSCs) are known to contribute to the progression of colorectal cancer (CRC). However, understanding of the molecular mechanisms and key factors involved in CRC is still insufficient to identify therapeutic targets against colorectal CSCs. In an effort to identify such mechanisms, we conducted bioinformatics analyses to evaluate the expression patterns in tumor and normal colorectal tissues, leading us to focus on the role of the ZNF217/Notch1 axis in mediating stem cell properties in CRC. Our findings revealed that ZNF217 overexpression activated self-renewal ability, expression of colorectal CSC markers, and Notch signaling in CRC. Dual-luciferase reporter assay suggested a role for ZNF217 in targeting Notch1 to activate Notch signaling. We observed that the promotional effects of Notch signaling, as well as CSC markers, under ZNF217 overexpression were attenuated after Notch1 knockdown. In addition to in vitro data, our in vivo results confirmed the inhibitory effect of sulforaphane on the tumorigenicity of CSCs, depicted the suppressive role of sulforaphane on colorectal CSCs mediated by the ZNF217/Notch1 axis, thereby providing new targetable vulnerabilities and therapeutic strategies for CRC.

Self-Renewal Inhibition in Breast Cancer Stem Cells: Moonlight Role of PEDF in Breast Cancer.

Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies have increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer-initiating cells and slow cellular cycle cells, identified by their capacity to retain long labeling (LT+). In this study, we perform new assays showing how stem cell self-renewal modulating proteins, such as PEDF, can modify the properties, percentage of biomarker-expressing cells, and carcinogenicity of cancer stem cells. The PEDF signaling pathway could be a useful tool for controlling cancer stem cells' self-renewal and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells' in vitro culture. We have designed a peptide consisting of the C-terminal part of this protein, which acts by blocking endogenous PEDF in cell culture assays. We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this modified PEDF protein produces a significant decrease in the percentage of expressed cancer stem cell markers.

Effect of Matrix Stiffness and Hepatocyte Growth Factor on Small Cell Lung Cancer Cells in Decellularized Extracellular Matrix-Based Hydrogels.

Small cell lung cancer (SCLC) is one of lethal cancers resulting in very low 5-year-survival rate. Although its clinical treatment largely relies on chemotherapy, SCLC cell physiology in three-dimenstional (3D) matrix has been less explored. In this work, the tumor microenvironment is reconstructed with decellularized porcine pulmonary extracellular matrix (dECM) with hyaluronic acid. To modulate matrix stiffness, the methacrylate groups are introduced into both dECM and hyaluronic acid, followed by photocrosslinking with photoinitiator. The stiffness of the resulting dECM-based hydrogel covers the stiffness of normal or cancerous tissue with varying dECM content. The proliferation and cancer stem cell marker expression of encapsulated SCLC cells are promoted in a compliant hydrogel matrix, which has a low shear modulus similar to that of the normal tissue. The hepatocyte growth factor (HGF) that induces SCLC cell invasion and chemoresistance markedly increases invasiveness and gene expression levels of CD44 and Sox2 in the hydrogel matrix. In addition, HGF treatment causes higher resistance against anticancer drugs (cisplatin and paclitaxel) in the 3D microenvironment. These findings indicate that malignant SCLC can be recapitulated in a pulmonary dECM-based matrix.

Kindlin-2 regulates colonic cancer stem-like cells survival and self-renewal via Wnt/β-catenin mediated pathway

BackgroundCancer Stem Cells (CSCs) have emerged as a critical mediator in recurrence and resistance in cancers. Kindlin-isoform (1 and 2) binds with cytoplasmic β-tail of integrin and are essential co-activators of integrin function. Given their important function in regulating cancer hallmarks such as cell proliferation, invasion, migration, and metastasis, we hypothesize that it might play a critical role in CSC growth, survival, and self-renewal of colon cancer. Materials and methodsUsing knockdown approaches, we inhibited Kindlin-2 expression in HCT116 and HT29 colon cancer cells. Extreme limiting dilution and self-renewal assay were performed to measure the role of Kindlin in colonic CSC. Standard methods such as qRT-PCR and western blotting were carried out to understand the signaling cascade by which Kindlin regulates CSC marker expression and downstream targets. ResultsOur data show isoform-specific upregulation of Kindlin-2 in colonic CSCs. The silencing of Kindlin-2 reduces colonosphere formation, decreases CSC size, and self-renewal marker genes such as CD-133, CXCR-4, LGR-5, and C-MYC. Kindlin-2 silencing reduces colonosphere proliferation, invasion, and migration of colonic CSCs. Mechanistically, Kindlin-2 silencing reduces the expression, and nuclear localization of β-catenin, and decreases β-catenin target genes such as C-MYC, cyclin D1, DKK-1, and Snail-1. ConclusionOur study delineates the isoform-specific activity of Kindlin-2 in regulating Colonic CSC. Isoform-specific targeting of Kindlin-2 may be a novel strategy to tackle this devastating disease.