Cancer Stem Cell Properties Research Articles

PPAR/PDK4 pathway is involved in the anticancer effects of cGMP in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with a high mortality rate. Current treatments for PDACs often have side effects, and drug resistance in cancer stem cells (CSCs) would be also a problem. Cyclic guanosine monophosphate (cGMP) suppresses the mitochondrial function of PDACs and inhibits their CSC properties. Metabolic regulation plays a crucial role in the maintenance of CSC phenotype, and we hypothesized that cGMP induction suppresses cancer stem cell properties in the cancer cell through energy-related signaling pathways. We demonstrated that induction of cGMP upregulated the PPARα/PDK4 pathway and suppressed CSC properties in PDAC, and patients with pancreatic cancer with high PDK4 gene expression had a better prognosis than those with low gene expression. Therefore, these mechanisms may provide new therapeutic targets for the eradication of pancreatic CSCs.

Cancer stem cell isolation and characterization from lung cancer cells

Lung cancer is one of the most common cancers in the world and a leading cause of death. Cancer stem cells (CSCs) are the cells responsible for tumor initiation. CSCs features include self-renewal capacity, differentiation, high invasion-migration, and resistance to chemotherapy. CSCs are characterized by specific surface markers. CD133 is widely used as a marker of CSCs in many cancers, including lung cancer. CD133-positive cells are an important marker of lung cancer stem cells because they show characteristics of lung cancer CSCs. CSCs are less sensitive and/or resistant to conventional treatments, remaining viable and regenerating tumors after treatment. Treatments that can target CSCs will be able to destroy CSCs more effectively, leading to the disappearance of tumors. Currently, attempts are being made to develop therapeutics that can effectively target CSCs. These studies concern the isolation and characterization of CSCs. In this study, CD133-positive CSCs and CD133-negative cells were isolated from H460 cells, a large cell lung cancer model, by MACS method. CSCs properties were investigated by tumorSphere formation assay (2% agar plate), hanging drop assay and qPCR method. CD133 positivity of H460 cells was determined as 0.3%. It has been observed that CD133-positive cells show CSCs-specific self-renewal properties. It was determined that the CD133 gene expression difference between CD133-positive CSCs and CD133-negative cells was approximately 25-fold. It has been observed that CD133-positive CSCs express more pluripotency genes (such as OCT-4, SOX-2 and KLF-4) compared to CD133-negative cells.

A FOXC2 inhibitor, MC-1-F2, as a therapeutic candidate for targeting EMT in castration-resistant prostate cancer

Androgen deprivation therapy (ADT) is the major treatment option for advanced prostate cancer. However, prostate cancer can develop into androgen-independent castration-resistant prostate cancer (CRPC) which is resistant to ADT. An alternative treatment strategy for CRPC can be targeting the epithelial-mesenchymal transition (EMT). EMT is governed by a series of transcription factors of which forkhead box protein C2 (FOXC2) is a central mediator. Our previous research into the inhibition of FOXC2 in breast cancer cells lead to the discovery of MC-1-F2, the first direct inhibitor of FOXC2. In current study on CRPC, MC-1-F2 has shown a decrease in mesenchymal markers, inhibition of cancer stem cell (CSC) properties and decrease in invasive capabilities of CRPC cell lines. We have also demonstrated a synergistic effect between MC-1-F2 and docetaxel treatments, leading to a decrease in docetaxel dosage, suggesting the possible combination therapy of MC-1-F2 and docetaxel for the effective treatment of CRPC.

RAB6A functions as a critical modulator of the stem-like subsets in cholangiocarcinoma.

RAB6A is a member of RAB GTPase family and plays an important role in the targeted transport of neurotrophic receptors and inflammatory cytokines. RAB6A-mediated secretory pathway is involved in many physiological and pathological processes. Defects in RAB6A-mediated secretory pathway may lead to the development of many diseases, including cancer. However, its role in cholangiocarcinoma (CCA) has not yet been revealed. We explored the regulatory role of RAB6A in the stem-like subsets of CCA. We showed that RAB6A knockdown (KD) impedes cancer stem cells (CSCs) properties and epithelial-mesenchymal transition in vitro and that suppression of RAB6A inhibits tumor growth in vivo. We screened target cargos of RAB6A in CCA cells and identified a extracellular matrix component as the target cargo. RAB6A binds directly to OPN, and RAB6A KD suppressed OPN secretion and inhibited the interaction between OPN and αV integrin receptor. Moreover, RAB6A KD inhibited the AKT signaling pathway, which is a downstream effector of the integrin receptor signaling. In addition, shRNA targeting OPN blocked endogenous expression of OPN and consequently weakened CSCs properties in RAB6A-formed spheres. Similarly, inhibitor of AKT signaling, MK2206 also impedes oncogenic function of RAB6A in the stem-like subsets of CCA cells. In conclusion, our findings showed that RAB6A sustains CSCs phenotype maintenance by modulating the secretion of OPN and consequentially activating the downstream AKT signaling pathway. Targeting the RAB6A/OPN axis may be an effective strategy for CCA therapy.

Abstract A068: Prostate luminal progenitors as the cell-of-origin for androgen receptor-independent prostate cancer

Abstract In the recent years, we have witnessed the emergence of androgen receptor (AR)-independent prostate cancer (AIPC) with the clinical use of second-generation androgen deprivation therapy, namely Enzalutamide and Abiraterone. Unluckily, treatment options for the end-stage AIPC patients are mainly palliative but not curable. However, the cellular origin and mechanisms involved in the evolution of AIPCs remain unclear. Previously, we have discovered that AR is not required for the survival and maintenance of progenitor property of CA stration- R esistant N kx3.1-expressing cells (CARNs), thus highlighting the AR-independent nature of this luminal progenitor subset. Interestingly, AR-deleted CARNs demonstrate gene expression profile that is highly similar to human neuroendocrine prostate cancer (NEPC), a subtype of AIPC. Moreover, upon Pten deletion and mutated Kras activation, AR-deleted CARNs can generate tumors exhibiting focal neuroendocrine differentiation, indicating the ability of CARNs to serve as a cell-of-origin for NEPC. Based on these observations, we hypothesize that AR-independent prostate luminal progenitor subsets are capable of initiating AIPC upon oncogenic transformation, while the transformed cells represent distinct subsets in AIPC that possess cancer stem cell property. In our latest assessments, we have validated the ability of CARNs to initiate NEPC using a novel genetically-engineered mouse model of prostate cancer. Moreover, we have established tumor organoids from CARNs-initiated AIPC, which are capable of generating allografts in both immunodeficient and immunocompetent mice, implying their immune-evading capability. Lastly, we have performed small molecule library screening on the transformed CARNs-derived tumor organoids to identify effective treatment options for AIPC. Taken together, our study has provided novel insights into the cellular origin of AIPCs. Consequently, targeting these prostate luminal progenitors-initiated tumor cells as well as their molecular property may serve as novel therapeutic strategies to overcome AR independence in prostate cancer. Citation Format: Chee Wai Chua. Prostate luminal progenitors as the cell-of-origin for androgen receptor-independent prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A068.

METTL3-mediated ALDH m6A methylation regulates the malignant behavior of BMI1+ HNSCC stem cells.

To reveal the effect and mechanism of methyltransferase-like 3 (METTL3) on cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC). First, we analyzed 14-HNSCC-patients' scRNA-seq dataset and TCGA dataset of HNSCC. Then, Mettl3 knockout or overexpression mice models were studied via tracing and staining technologies. In addition, we took flow cytometry sorting and sphere formation assays to observe tumorigenicity and used cell transfection and western blotting to verify target protein expression levels. Furthermore, methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative real-time PCR (MeRIP-qPCR) were taken to identify the mechanism of Mettl3 regulating Bmi1+ CSCs in HNSCC. Due to SOX4 transcriptional regulation, METTL3 regulated the malignant behavior of BMI1+ HNSCC stem cells through cell division pathway. The progression and malignancy of HNSCC were decreased after Mettl3 knocked-out, while increased after Mettl3 knocked-in in Bmi1+ CSCs in vivo. Knockdown of Mettl3 inhibited stemness properties of CSCs in vitro. Mechanically, Mettl3 mediated the m6A modification of ALDH1A3 and ALDH7A1 mRNA in Bmi1+ HNSCC CSCs. Regulated by SOX4, METTL3-mediated ALDH m6A methylation regulates the malignant behavior of BMI1+ HNSCC CSCs through cell division pathway.

High expression of PKCλ and ALDH1A3 indicates a poor prognosis, and PKCλ is required for the asymmetric cell division of ALDH1A3-positive cancer stem cells in PDAC

Pancreatic ductal adenocarcinoma (PDAC) is the cancer with the poorest prognosis. One of the major properties reflecting its poor prognosis is high-grade heterogeneity, which leads to insensitivity to anticancer treatments. Cancer stem cells (CSCs) acquire phenotypic heterogeneity, generating abnormally differentiated cells by asymmetric cell division. However, the detailed mechanism leading to phenotypic heterogeneity is largely unknown. Here, we showed that PDAC patients with co-upregulation of PKCλ and ALDH1A3 had the poorest clinical outcome. PKCλ knockdown by DsiRNA in the ALDH1high population of PDAC MIA-PaCa-2 cells attenuated the asymmetric distribution of the ALDH1A3 protein. To monitor asymmetric cell division of ALDH1A3-positive PDAC CSCs, we established stable Panc-1 PDAC clones expressing ALDH1A3-turboGFP (Panc-1-ALDH1A3-turboGFP cells). In addition to MIA-PaCa-2-ALDH1high cells, turboGFPhigh cells sorted from Panc-1-ALDH1A3-turboGFP cells showed asymmetric cell propagation of ALDH1A3 protein. PKCλ DsiRNA in Panc-1-ALDH1A3-turboGFP cells also attenuated the asymmetric distribution of ALDH1A3 protein. These results suggest that PKCλ regulates the asymmetric cell division of ALDH1A3-positive PDAC CSCs. Furthermore, Panc-1-ALDH1A3-turboGFP cells can be useful for the visualization and monitoring of CSC properties such as asymmetric cell division of ALDH1A3-positive PDAC CSCs in time-lapse imaging.

Targeting Pokemon is a novel strategy to suppress cancer aggressiveness of non-small cell lung cancer: Identification of Pokemon as ideal target for developing anti-NSCLC drugs

Although it is widely reported that Pokemon acts as an oncogene in the pathogenesis of multiple cancers, but its role and detailed molecular mechanisms in regulating non-small cell lung cancer (NSCLC) progression have not been fully delineated. Here, by performing Real-Time qPCR analysis, we verified that Pokemon was high-expressed in NSCLC tissues and cells, compared to the corresponding normal lung tissues and epithelial cells. Then, the small interfering RNA (siRNA) for Pokemon was transfected into the NSCLC cells to verify its biological functions, and our results suggested that silencing of Pokemon suppressed the malignant phenotypes, including cell viability, mitosis, colony formation, epithelial-mesenchymal transition (EMT), mobility and cancer stem cell (CSC) properties in NSCLC cells. Mechanistically, we confirmed that knockdown of Pokemon decreased the expression levels of phosphorylated Akt (p-Akt), phosphorylated GSK-3β (p-GSK-3β) and Snail to inactivate the oncogenic Akt/GSK-3β/Snail signal pathway, and deletion of Snail also had similar effects to hamper the development of NSCLC. Next, our rescuing experiments validated that Pokemon ablation-induced suppressing effects on NSCLC cell malignancy were all abrogated by overexpressing Snail. Finally, the in vivo experiments confirmed that silencing of Pokemon downregulated Snail to hamper tumorigenesis of NSCLC cells in xenograft tumor-bearing mice models. Taken together, we firstly uncovered the underlying mechanisms by which the Pokemon/Akt/GSK-3β/Snail signal pathway contributed to the development of NSCLC, and this signal pathway could be potentially used as therapeutic targets for the development of personalized anti-NSCLC drugs.

TMT-based quantitative proteomic analysis of spheroid cells of endometrial cancer possessing cancer stem cell properties

BackgroundCancer stem cells (CSCs) play an important role in endometrial cancer progression and it is potential to isolate CSCs from spheroid cells. Further understanding of spheroid cells at protein level would help find novel CSC markers.MethodsSpheroid cells from endometrial cancer cell lines, Ishikawa and HEC1A, exhibited increased colony forming, subsphere forming, chemo-drug resistance, migration, invasion ability and tumorigenicity, verifying their cancer stem-like cell properties. The up-regulated CD90, CD117, CD133 and W5C5 expression also indicated stemness of spheroid cells. TMT-based quantitative proteomic analysis was performed to explore the potential alterations between parent cells and cancer stem-like spheroid cells. HK2-siRNA was transfected to Ishikawa and HEC1A cells to explore the roles and molecular mechanism of HK2 in endometrial cancer.ResultsWe identified and quantified a total of 5735 proteins and 167 overlapped differentially expressed proteins of two cell types, 43 proteins were up-regulated and 124 were down-regulated in spheroid cells comparing with parent cells. KEGG pathway revealed a significant role of HIF-1 pathway in spheroid cells. qRT-PCR and western blot results of GPRC5A, PFKFB3 and HK2 of HIF-1 pathway confirmed their elevated expressions in spheroid cells which were consistent with proteomic results. HK2 promoted cancer stemness in endometrial cancer.ConclusionThese findings indicate that spheroid cells from endometrial cancer cell lines possess cancer stem-like cell properties and enrich CSCs. HIF-1 pathway is activated in endometrial cancer stem-like spheroid cells.

Evaluation of the Effectiveness of Various Autophagy Inhibitors in A549 Cancer Stem Cells.

Numerous studies have already established that autophagy plays a central role in the survival of all cells, including malignant ones. Autophagy is a central cog in the general mechanism that provides the intracellular proteostasis determining cellular physiological and phenotypic characteristics. The accumulated data show that autophagy largely contributes to cancer cell stemness. Thus, autophagy modulation is considered one of the promising pharmacological targets in therapy aimed at cancer stem cell elimination. However, autophagy is a multi-stage intracellular process that involves numerous protein participants. In addition, the process can be activated simultaneously by various signaling modules. Therefore, it is no small feat to select an effective pharmacological drug against autophagy. What's more, the search for potential chemotherapeutic agents that could eliminate cancer stem cells through pharmacological inhibition of autophagy is still under way. In the present work, we selected a panel of autophagy inhibitors (Autophinib, SBI-0206965, Siramesine, MRT68921, and IITZ-01), some of whom have been recently identified as effective autophagy inhibitors in cancer cells. Using A549 cancer cells, which express the core stem factors Oct4 and Sox2, we evaluated the effect of these drugs on the survival and preservation of the original properties of cancer stem cells. Among the agents selected, only Autophinib demonstrated a significant toxic effect on cancer stem cells. The obtained results demonstrate that autophagy inhibition by Autophinib downregulates the expression of the Sox2 protein in A549 cells, and that this downregulation correlates with a pronounced induction of apoptosis. Moreover, Autophinib-treated A549 cells are unable to form spheroids, which indicates a reduction in stemness. Thus, among the drugs studied, only Autophinib can be considered a potential agent against cancer stem cells.

Amino acid profile changes during enrichment of spheroid cells with cancer stem cell properties in MCF-7 and MDA-MB-231 cell lines.

Cancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis. This study aimed to evaluate the changes in the amino acid profile in MCF-7 and MDA-MB-231 cells during spheroid formation to discover the specific metabolic properties in CSCs. MCF-7 and MDA-MB-231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high-performance liquid chromatography (HPLC). The results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF-7 and MDA-MB-231 spheroids (histidine and serine, respectively). Our results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation.

FAK downregulation suppresses stem-like properties and migration of human colorectal cancer cells.

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase, which is overexpressed in colorectal cancer cells. FAK could be activated by phosphorylation to participate in the transduction of multiple signaling pathways and self-renewal of cancer stem cells. Whether the downregulation of FAK inhibits the metastasis in colorectal cancer through the weakening of stem cell-like properties and its mechanisms has yet to be established. CD44, CD133, c-Myc, Nanog, and OCT4 were known to mark colorectal cancer stem cell properties. In this study, AKT inhibitor (MK-2206 2HCl) or FAK inhibitor (PF-562271) decreased the expression of stem cell markers (Nanog, OCT4, CD133, CD44, c-Myc) and spheroid formation in colorectal cancer. Moreover, FAK and AKT protein was shown to interact verified by co-immunoprecipitation. Furthermore, downregulation of FAK, transfected Lenti-FAK-EGFP-miR to colorectal cancer cells, reduced p-AKT but not AKT and decreased the expression of stem cell markers and spheroid formation in colorectal cancer. In conclusion, we demonstrated that downregulation of FAK inhibited stem cell-like properties and migration of colorectal cancer cells partly due to altered modulation of AKT phosphorylation by FAK.

Metastatic niches in patients with high-grade neuroendocrine tumours: Apparent findings of matched triple-positive radiolabelled molecular probes by nuclear medicine sequence imaging

Nuclear medicine imaging of neuroendocrine neoplasm (NEN) patients could evidence cancer cell states related to glycolytic hypermetabolism, somatostatin (SST) receptors overexpression or norepinephrine (NE) transport system hyperactivity status of cancer cells profiles, according to the radiolabelled molecular probe used: fluorodeoxyglucose (FDG), octreotide (SST analogue) or the compound metaiodobenzylguanidine (NE analogue), respectively. NEN lesions with positive positron emission tomography with 18FDG (glucose analogue radiolabelled with fluoride-18) stablished a relationship with tumour aggressiveness and poorly differentiated cell morphology. Otherwise, NEN lesions evidencing somatostatin receptors (SSTR) overexpression or abnormal surface NE transporter system hyperactivity status of cancer cell profiles correlated with well differentiated cell morphology. Theoretically, the apparent correspondence between the triple-positive imaging of glycolytic hypermetabolism, SSTR overexpression and abnormal NE transporter system hyperactivity of overlapping cancer cell states in metastatic site of a NEN patient could be revealing a single tumorigenic subpopulation with a dynamic divergent differentiation potential. The complex events of metastasis progression included transdifferentiation from epithelial-to-mesenchymal to leave the primary neoplastic niche, probable as a collective migration of cancer cells, including cancer stem cells (CSCs), to circulating and reversibly transdifferentiating from mesenchymal-to-epithelial to settle in distant niches. Considering future research perspectives, we argued whether the apparent triple-positive matched correlative imaging with the radiolabelled molecular probes (glucose, SST and NE analogues) in patients’ metastatic niches could indicate potential biopsy sites to further investigation for the potential CSCs properties. We suggested that early treatment planning with lutetium-177-based peptide receptor radionuclide therapy, could provide for the blocking of poorly differentiated cell states at the point of differentiation to well differentiated cell states, and vice versa. In addition, specific individual cases should be considered for targeted-based therapy with radiolabelled-NE analogue, blocking dedifferentiation and transdifferentiation from abnormal NE transporter system hyperactivity status to SSTR overexpression cancer cell profile.

Effective inhibition of breast cancer stem cell properties by quercetin-loaded solid lipid nanoparticles via reduction of Smad2/Smad3 phosphorylation and β-catenin signaling pathway in triple-negative breast cancer

BackgroundThe presence of cancer stem cells (CSCs) is a major cause of resistance to cancer therapy and recurrence. Triple-negative breast cancer (TNBC) is a subtype that responds poorly to therapy, making it a significant global health issue. Quercetin (QC) has been shown to affect CSC viability, but its low bioavailability limits its clinical use. This study aims to increase the effectiveness of QC in inhibiting CSC generation by using solid lipid nanoparticles (SLNs) in MDA-MB231 cells. Materials and methodsAfter treating MCF-7 and MDA-MB231 cells with 18.9 μM and 13.4 μM of QC and QC-SLN for 48 h, respectively, cell viability, migration, sphere formation, protein expression of β-catenin, p-Smad 2 and 3, and gene expression of EMT and CSC markers were evaluated. ResultsThe QC-SLN with particle size of 154 nm, zeta potential of −27.7 mV, and encapsulation efficacy of 99.6% was found to be the most effective. Compared to QC, QC-SLN significantly reduced cell viability, migration, sphere formation, protein expression of β-catenin and p-Smad 2 and 3, and gene expression of CD44, zinc finger E-box binding homeobox 1 (ZEB1), vimentin, while increasing the gene expression of E-cadherin. ConclusionsOur findings demonstrate that SLNs improve the cytotoxic effect of QC in MDA-MB231 cells by increasing its bioavailability and inhibiting epithelial-mesenchymal transition (EMT), thereby effectively inhibiting CSC generation. Therefore, SLNs could be a promising new treatment for TNBC, but more in vivo studies are needed to confirm their efficacy.

Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10-9 and 3.2 × 10-9 M, respectively. C44Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.

Abstract 6705: Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is a clinically challenging disease that requires a multidisciplinary approach. Unfortunately, the scarcity of ESCC genomic data hinders the understanding of ESCC biology, disease progression and rational therapy design. Circulating tumor cells (CTCs), which are shed from the primary and metastatic tumors and then circulate within the peripheral vasculature, reflect the existing tumor heterogeneity. Here, we utilized the non-biased size-based CTC enrichment strategy in combination with the real-time RT-PCR to molecularly characterize the CTCs with cancer stem cell (CSC) or mesenchymal properties in ESCC. Comprehensive data from ESCC cell lines, mouse ESCC xenograft models and clinical ESCC peripheral blood samples emphasize the importance of TWIST1 (Twist Family BHLH Transcription Factor 1) in ESCC progression. Gain-of-function and loss-of-function analyses demonstrate that TWIST1 promotes cell migration, invasion and colony formation in ESCC cells. Moreover, positive TWIST1 expression is responsible for the chemoresistance of ESCC cells to Cisplatin and is correlated with poor overall survival in ESCC patients. In addition, we show that TWIST1 promotes malignant potential, including tumor growth, invasion and chemoresistance via the TWIST1-TGFBI-ZEB1 signaling pathway in ESCC. Meanwhile, the TWIST1-TGFBI-ZEB1 signaling pathway confers immunosuppressive conditions to the tumor microenvironment, which in turn contributes to EMT to promote tumor progression. Collectively, these findings indicate that TWIST1 has a novel role in ESCC carcinogenesis by regulating tumorigenicity and cancer stem cell properties that may be useful as a prognostic monitoring biomarker and a promising therapeutic target in clinical ESCC treatment. Citation Format: Zhen Tan, Josephine Mun-Yee Ko, Valen Zhuoyou Yu, Hoi Yan Ng, Simon Law, Maria Li Lung. Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6705.

Abstract 488: New therapeutic strategies suppressing NAD+ and ATP production in cancer metabolism specific to 3D cultured ovarian cancer cells

Abstract Ovarian cancer is the gynecological cancer with the highest mortality rate. As resistance to treatment is acquired and many patients relapse, new treatment strategies are needed. A model that strongly reflects the features of recurrent ovarian cancer in the peritoneal cavity is the "cancer stem cells (CSCs)," which are enriched when cells are cultured in three dimensions (3D) on low-attachment plates. The unique metabolic system of the "3D CSCs" is the subject of a novel therapeutic approach. Here, we show that ovarian cancer 3D CSCs have strong NAD+ production capacity, which is particularly dependent on nicotinamide phosphoribosyl transferase (NAMPT), known as the rate-limiting enzyme in the salvage pathway of NAD+ production. We found that NAMPT inhibition blocks NADPH and ATP production as well as NAD+, and subsequently suppresses the synthesis capacity of proteins, fat droplets, and cholesterol in the 3D ovarian cancer cells. In addition, we found that the combination of NAMPT inhibitor and the ALDH inhibitor disulfiram had a pronounced synergistic effect. This synergy inhibits oxidative phosphorylation and ALDH activity and promotes reactive oxygen species and apoptosis via caspase 3/7 cleavage, which leads to death of the 3D cultured cells. Our results demonstrate that high NAMPT expression in ovarian cancer is likely to be a strong poor prognostic factor and that the combination of a NAMPT inhibitor and disulfiram could be a new treatment option to treat or prevent recurrent ovarian cancer. Our novel findings focus on NAMPT addiction as a property of ovarian cancer 3D CSCs, and strongly suggest the combined inhibition of NAMPT and ALDH as a potential therapeutic strategy. Citation Format: Kei Kudo, Brittney S. Harrington, Franklin Ning, Rahul Kamdar, Yusuke Shibuya, Soumya Korrapati, Maria Fergusson, Calen Kucera, Fantini Massimo, Christina M. Annunziata. New therapeutic strategies suppressing NAD+ and ATP production in cancer metabolism specific to 3D cultured ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 488.

Abstract 2443: Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the common malignancies worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and therapeutic resistance. Increasing evidence showed the existence of cancer stem cells (CSCs) which is crucial for tumor initiation and therapeutic resistance. However, there are limited kinase inhibitors available that show specific targeting against liver T-ICs for HCC treatment. To understand the mechanism for regulation of liver CSCs, we have employed RNA-Seq profiling to compare the gene expression profiles with focuses on protein kinases between (1) sorted CD133+ liver CSCs and CD133- differentiated counterparts isolated from two HCC cell lines (Huh7 and PLC8024) and cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. This analysis revealed Serine/Threonine kinase 39 (STK39), also known as SPAK, to be the commonly up-regulated protein kinase common in both CD133+ liver CSCs and CSC enriched hepatospheres. Consistently, based on the TCGA data analysis, STK39 mRNA expression is positively correlated with the expression of liver CSC markers including CD24, CD133, and CD47. Using publicly available transcriptome datasets deposited in the NCBI Gene Expression Omnibus (GEO) including GSE14520 and GSE25097, we found that overexpression of STK39 was also observed in HCC tumor tissues (p<0.001) and correlated with poorer overall survival and disease-free survival. By lentiviral based overexpression and knockdown approaches, we demonstrated the role of STK39 in regulation of liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness and expression of liver CSC markers. Specially, STK39 expression was highly elevated in sorafenib- and lenvatinib-resistant HCC cells, and its inhibition led to sensitization of HCC cells towards these treatments. In order to identify downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. Upon analysis, PARP1, was identified to be the novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, phosphorylation of PARP1 at Threonine 368 (T368) was found to be highly inhibited, suggesting its role as downstream effector of STK39-mediated liver CSC functions. Furthermore, STK39 regulates PARP1-mediated chromatin decondensation, and thus opens chromatin structure at the promoter regions of SOX2/OCT4. In summary, STK39 regulates cancer stemness and drug resistance by directly modulating PARP1-mediated SOX2/OCT4 expression via chromatin remodelling. Targeting STK39/PARP1 signaling cascade alone or in combination with molecular targeted drugs including rucaparib and lenvatnib can be the potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2443.

Abstract 1489: Overexpression of ribosomal L22-like1 (RPL22L1) enhances cancer stem cell properties and promotes tumor progression in hepatocellular carcinoma (HCC)

Abstract The abnormality of ribosomal biogenesis is found to be involved in carcinogenesis and progression in various types of cancer. RPL22L1 gene is a highly homologous paralog of RPL22, both of which belong to the L22E family of ribosomal proteins and take part in encoding a cytoplasmic ribosomal protein that is a component of the 60S subunit of ribosomes. Our previous work has established a CRISPR/Cas9 genome-wide knockout screening profile and developed a two-component mixture model to identify cancer-specific essential genes in 436 cancer cell lines. Bioinformatics analysis reveals that RPL22L1 acts as a feature gene to predict cancer-specific essential genes in human cancer. Previous studies suggest that RPL22L1 is correlated with tumor progression and adverse prognosis in several cancer types, while its role in hepatocellular carcinoma (HCC) is unknown. In the present study, we found that RPL22L1 is highly expressed in embryonic stem cells (ES) and liver progenitor cells, which were acquired from a vitro hepatic differentiation model originating from ES cells. Hence, we hypothesized that RPL22L1 enhances cancer stem cell properties in HCC. Functional assays showed that RPL22L1 would promote sphere formation in vitro. qRT-PCR and Western Blotting results indicated that RPL22L1 could promote stemness-related gene expressions such as CD133, CD44, and SOX9 in HCC cell lines. Overexpression of RPL22L1 increased aldehyde dehydrogenase (ALDH) activity, which was decreased when RPL22L1 was silenced with short hairpin RNA (shRNA). Extreme limiting dilution assay showed that RPL22L1 overexpression stimulated self-renewal ability in immunodeficient mice. Furthermore, higher RPL22L1 expression can also promote cell proliferation and epithelial-mesenchymal-transition, as well as drug resistance to 5-Fluorouracil (5-FU) and cisplatin (CDDP). Silver nitrate staining demonstrated that RPL22L1 overexpression correlated with a larger size and number of nucleoli. The expression of RPL22L1 was also associated with adverse prognosis in HCC clinical samples. In conclusion, our study reveals aberrant ribosomal protein activity in HCC and the role of RPL22L1 in promoting HCC tumor progression. Citation Format: JinLin Huang, Zhenyang Guo, Jie Luo, Beilei Liu, Lanqi Gong, Xiaona Fang, Dora Lai-wan Kwong, Xinyuan Guan. Overexpression of ribosomal L22-like1 (RPL22L1) enhances cancer stem cell properties and promotes tumor progression in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1489.

Abstract 269: New metabolic biomarker of ALDOA induces tumor aggressiveness in breast cancer and nicotinamide as a potential anti-tumor agent targeting ALDOA

Abstract Introduction: Nicotinamide (NAM), a water-soluble form of niacin (Vitamin B3), is a precursor of nicotinamide-adenine dinucleotide (NAD+) and regulates cellular energy metabolism. In our previous study with metabolic RNA panel assay, we confirmed ALDOA (Aldolase A) is suppressed after treatment of nicotinamide in breast cancer organoids. ALDOA is a crucial glycolytic enzyme that turns fructose-1,6-bisphosphate (FBP) into glyceraldehyde-3-phosphate. Several studies have shown that high expression of ALDOA is associated with poor prognosis in various types of cancer. However, its function in breast cancer remains unclear. Methods: ALDOA expression in breast cancer was analyzed with METABRIC data set. A total of 79 fresh cancer tissues and tumor microarrays (TMA) with 402 breast cancer patients were enrolled. Metabolic RNA sequencing panel assay was performed with human breast cancer organoids after treatment of nicotinamide. The breast cancer cell lines including MCF7, T47D, and BT474 were generated to establish stable cells in which ALDOA expression is continuously reduced using lentiviral system. Total RNA was isolated from human breast cancer cells and performed RT-PCR for Sanger sequencing. Proliferation assay was performed using cell counting kit-8. We used transwell migration and invasion assay. To identify cancer stemness, we performed sphere formation assay. Mitochondrial function was measured using the Seahorse XF24 Flux Analyzer. Results: In our previous study, after the treatment of nicotinamide, ALDOA expression in breast cancer organoids was suppressed. So we assumed that ALDOA has an oncogenic function in cellular metabolism. The expression of ALDOA was much higher in breast cancer clinical tissues compared with normal tissues. High expression of ALDOA was correlated with the worse prognosis of breast cancer patients in METABRIC public data and our own cohort. Sanger sequencing in the cell lines showed no mutation in exon5 to exon9 which is known as mutation hotspot in breast cancer. Cell proliferation activity and ability of cellular movement were inhibited in ALDOA-downregulated breast cancer cells. In the sphere formation assay, we found that the tumor cells with low expression of ALDOA decreased cancer stem cell properties. When POU2F1 or POU2F2 was suppressed, ALDOA protein expression was also inhibited. ECAR and OCR concerning mitochondrial metabolic functions were also downregulated in the tumor cells with ALDOA suppression. Conclusions: In conclusion, we suggest that ALDOA might be a key biomarker inducing tumor aggressiveness by modulating metabolic processes in breast cancer. Our results suggest that nicotinamide can be a novel therapeutic candidate targeting in breast cancer with high expression of ALDOA. Citation Format: Da Sol Kim, Cheng Hyun Lee, Soo Young Park, Han Suk Ryu. New metabolic biomarker of ALDOA induces tumor aggressiveness in breast cancer and nicotinamide as a potential anti-tumor agent targeting ALDOA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 269.