Stemness Properties Research Articles

Single-cell transcriptomics reveals the intra-tumoral heterogeneity and SQSTM1/P62 and Wnt/β-catenin mediated epithelial to mesenchymal transition and stemness of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by the complex tumor microenvironment (TME) consisting of an abundance of mesenchymal stem cells (MSCs), which is known to facilitate epithelial-to-mesenchymal transition (EMT). The development of single-cell genomics is a powerful method for defining the intricate genetic landscapes of malignancies. In this study, we have employed single-cell RNA sequencing (scRNA-seq) to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare consequential cell subpopulations of significance. The scRNA-seq analysis of TNBC and Normal patient derived samples revealed that EMT markers and transcription factors were most upregulated in MSC population. Further, exploration of gene expression analysis among TNBC and Normal patient-derived MSCs ascertained the role of SQSTM1/P62 and Wnt/β-catenin in TNBC progression. Wnt/β-catenin and Wnt/PCP signaling pathways are prominent contributors of EMT, stemness, and cancer stem cell (CSC) properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT.Moreover, siRNA targeting SQSTM1/P62 and inhibitor of Wnt/β-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis. The synergistic impact of inhibition of SQSTM1/P62 and Wnt/β-catenin signaling facilitates the development of a potential therapeutic regimen for TNBC.

Abstract SY34-01: Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy

Abstract Cellular plasticity and stemness properties enable dynamic changes of circulating tumor cells (CTCs) during cancer dissemination, such as aggregation or cohesion of single CTCs into multicellular CTC clusters with 20-100 times higher metastatic propensity than the singles. Our work demonstrated that stemness glycoproteins on breast cancer stem cells, such as CD44, CD81, and ICAM1 drive CTC aggregation in metastatic breast cancer, such as triple negative breast cancer (TNBC). To determine the glycosylation patterns in CTCs and its association with clinical outcomes, we established multiple CTC analysis approaches including CellSearch, flow cytometry, and immunohistochemistry using patient blood and tissue sections collected at the pre-treatment baseline and after-therapy timepoints. We found that chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of terminal sugar residues α2,6-sialic acids in glycoproteins. CTCs showed dynamic hypo-sialylation in the blood with loss of the sialyl-transferase ST6GAL1, promoting CTC cluster formation with cellular quiescence (proliferative dormancy) and evading chemotherapy in breast cancer. Seeded tumor cells regained ST6GAL1 to enable metastatic colonization. Many adhesion proteins and stemness drivers as glycoprotein substrates of ST6GAL1 drive CTC clustering and metastatic seeding. Neutralizing antibodies against these clustering drivers inhibit CTC cluster formation and improve therapy response, thereby blocking lung metastasis in TNBC. Citation Format: Huiping Liu. Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY34-01.

Does lower water availability limit stem CO2 efflux of oak and hornbeam coppices?

Recent changes in water availability can be crucial for the development, growth and carbon budget of forests. Therefore, our aim was to determine the effect of reduced throughfall and severe summer drought on stem CO2 efflux as a function of temperature and stem increment. Stem CO2 efflux was measured using the chamber method on oak and hornbeam under four treatments: coppice, thinned coppice, and both coppice and thinned coppice with 30 %-reduced throughfall. The first year of the experiment had favourable soil water availability and the second year was characterized by a dry summer. While reduced throughfall had no effect on stem CO2 efflux, the summer drought decreased efflux by 43-81 % during July and August. The stem CO2 efflux was reduced less severely (by 13-40 %) in September when the drought persisted but the stem increment was already negligible. The stem increment was also strongly affected by the drought, which was reflected in its paired relationship with stem CO2 efflux over the two experimental years. The study showed that summer dry periods significantly and rapidly reduce stem CO2 efflux, whereas a constant 30 % rainfall reduction needs probably a longer time to affect stem properties, and indirectly stem CO2 efflux.

The outcome of tissue cryopreservation on the cellular, molecular and epigenetic characteristics of endometrial tissue and stromal cells

Research questionWhat impact does the cryopreservation of endometrial tissue have on cell characteristics and molecular and epigenetic profile changes in endometrial tissue and stromal cells? DesignCellular properties, such as proliferation efficiency, surface marker expression and the differentiation potency of endometrial stromal cells (ESC) isolated from fresh (Native) and cryopreserved (Cryo) tissue were compared. Moreover, changes in the expression of genes associated with pluripotency, endometrial function and epigenetic regulation and microRNA (miRNA, miR) were assessed, as were levels of DNA methylation and histone modifications. ResultsNative and Cryo cells exhibit very similar profiles including cell surface marker expression, differentiation potency and histone modifications, except for a decrease in proliferative potency and cell surface marker SUSD2 expression in Cryo cells. It was demonstrated that endometrial tissue cryopreservation led to an up-regulated expression of genes associated with pluripotency (NANOG, OCT4 [also known as POU5F1]). This confirms that despite being recovered from cryopreserved differentiated tissue, cells retained their stemness properties. In addition, alterations in DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) gene regulation were observed, along with a down-regulation of hsa-miR145-5p in Cryo ESC. ConclusionsThese findings contribute to a deeper understanding of the complex effects of endometrial tissue cryopreservation, providing insights for both medical and basic research applications. Since different tissues possess unique characteristics, it is essential to select the most suitable cryopreservation method for each tissue individually. Furthermore, the study findings indicate the potential utility of slow-cooling cryopreservation for both normal and pathological endometrial tissue samples, with the purpose of isolating stromal cell cultures.

Optimization of the Amplification of Equine Muscle-Derived Mesenchymal Stromal Cells in a Hollow-Fiber Bioreactor.

The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull's eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications.

Optimal Treatment Parameters for Ultrasound-Stimulated Microbubbles in Upregulating Proliferation and Stemness of Bone Marrow Mesenchymal Stem Cells.

Ultrasound-targeted microbubble disruption (UTMD) is a widely used technique to improve the differentiation and proliferation capacity of mesenchymal stem cells (MSCs), but the optimal therapeutic parameters for UTMD are unclear. In this study, we aimed to find the appropriate peak negative pressure (PNP), which is a key parameter for enhancing the stemness properties and proliferation of MSCs. Experiments were performed in UTMD group, ultrasound (US) group under different PNP exposure conditions (0.5, 1.0, and 1.5 MPa), and control group. Apoptosis safety was analyzed by flow cytometry and MSC proliferation was measured at 12, 24, and 36 hours after irradiation by cell counting kit 8. The expression of the stemness genes NANOG, OCT-4, and SOX-2 were determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction. The results showed that the 1.5 MPa UTMD-treated group had the highest proliferation capacity of MSCs at 24 hours. ELISA or quantitative reverse transcription polymerase chain reaction results showed that UTMD treatment of the 1.5 MPa group significantly upregulated the expression of the stemness genes NANOG, SOX-2, and OCT-4. In conclusion, the appropriate peak PNP value of UTMD was 1.5 MPa, and 1.5 MPa-mediated UTMD group obviously promoted MSCs proliferation and maintained stemness by upregulating the expression of stemness genes.

MiR-199a-5p targets DUSP14 to regulate cell proliferation, invasion and stemness in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) shows the highest morbidity among malignant tumors worldwide. Despite improvements of diagnosis and treatment, patient prognosis remains unfavorable. Therefore, there is a need to discover a novel treatment strategy for NSCLC. DUSP14 is related to various cancers as the regulatory factor for cellular processes. However, its specific roles in NSCLC and the upstream modulator remain largely unclear. MethodsDUSP14 expression patterns within the lung cancer patient cohort from TCGA database were analyzed using UALCAN online tool. Different databases including miRDB, starbase, and Targetscan were employed to screen the upstream regulator of DUSP14. DUSP14 and miR-199a-5p expression was determined by qRT-PCR and Western blot techniques. To confirm binding interaction of DUSP14 with miR-199a-5p, we conducted a dual-luciferase reporter assay. Cell viability, migration, and stemness properties were assessed using CCK-8, EdU (5-ethynyl-2′-deoxyuridine) incorporation, transwell invasion, and sphere formation assays. The effect of DUSP14 silencing on tumorigenesis was assessed with the NSCLC cell xenograft mouse model. ResultsOur study discovered that DUSP14 exhibited high expression within NSCLC tumor samples, which is related to the dismal prognostic outcome in NSCLC patients. Silencing DUSP14 impaired NSCLC cell proliferation, migration, and tumor sphere formation. Besides, we identified miR-199a-5p as the upstream regulatory factor for DUSP14, and its expression was negatively related to DUSP14 level within NSCLC tissues. Introducing miR-199a-5p recapitulated the function of DUSP14 silencing in NSCLC cell aggressiveness and stemness. Moreover, knocking down DUSP14 efficiently inhibited tumor formation in NSCLC cells of the xenograft model. ConclusionsOur study suggests that DUSP14 is negatively regulated by miR-199a-5p within NSCLC, whose overexpression is required for sustaining NSCLC cell proliferation, invasion and stemness.

Ethno‐Botanical Study of Medicinal Plants from Vidisha, Madhya Pradesh: Methanolic Extracts of Four Species and their Antimicrobial, Anti‐Inflammatory and Toxicity Properties

AbstractThe traditional usage of Plumbago zeylanica L, Cassia alata L, Caesalpinia crista L, and Rubia Cordifolia L in the villages of Vidisha district in Madhya Pradesh, India, has sparked research into the plants' phytochemical makeup and a range of possible medicinal benefits. This research is the first to describe the phytochemical makeup, powerful anti‐inflammatory, and antioxidant properties of PZ stem. The PZ stem and CA fruit methanolic extracts has the greatest levels of phenolic, flavonoid, and flavonol compounds and has a robust antioxidant capacity for scavenging DPPH and hydrogen peroxide with regard to A. flavus, A. baumannii, A. fumigatus, S. aureus, K. pneumonia, and R. oryzae, the anti‐bacterial and anti‐fungal activity of the chosen plant extracts are strongest for PZ, CA, and CA T. terrestris leaf.

Silica nanoparticles improve growth, chemical bioactive, and antioxidant enzyme activity of Dianthus caryophyllus L., plant

Background Nanotechnology has provided innovative use of various materials with unique properties. These substances may react positively in plants, and the induction of bioactive compounds and antioxidant activity is particularly remarkable. Ornamental plant production is a positive economic activity of great interest, especially Carnation (Dianthus caryophyllus L.). This plant is more attractive for its various colors, but the flower quality and its longevity can diminish very fast. Thus, examining methods capable of improving flower quality and vase life is crucial. Additionally, carnation can be used to treat coronary and nervous disorders. Carnation contains compounds that calm the nervous system and minimize swelling and inflammation. Objective This research aimed to evaluate the effect of silica nanoparticles (SiO2 NPs) on the growth, bioactive chemical analysis, enzyme activity, and antioxidant accumulation. Material and methods The different rates of SiO2 NPs (0, 150, and 300 ppm) were used in two ways: foliar spray and soil drench; it was applied 3 times. The first application was after 3 weeks of transplanting and repeated with 21-day intervals. Results and conclusion The results showed that foliar application of SiO2 NPs increased all morphological and flowering traits compared with drench and the control, higher levels of photosynthetic pigments and anthocyanin, greater total phenols, total sugars, and total free amino acids were obtained from plants treated with foliar spray 300 ppm followed than 150 ppm. In addition, the greatest levels of enzyme activity were recorded with plants treated with foliar spray of SiO2 NPs at a rate of 300 ppm, as well as improved anatomical structure of the stem and stomata properties compared with the drench method and control. Similarly, SiO2 NPs application method is more critical than their concentration. Therefore, it is recommended that foliar spraying of SiO2 NPs at 300 ppm can enhance plant growth, flowering, bioactive compounds, enzyme activity, and anatomical structure of the stem of carnation plants while adding SiO2 NPs drench at a rate of 150 ppm had rather acceptable findings.

SF1: A Standardised Fraction of Clinacanthus nutans That Inhibits the Stemness Properties of Cancer Stem-Like Cells Derived from Cervical Cancer

Cancer stem cells (CSCs) are a small population of tumour cells that are responsible for tumour initiation, metastases, recurrence, and resistance to conventional therapy. Hence, targeting CSCs is crucial in the fight against cancer. SF1, a standardised fraction from Clinacanthus nutans leaf extract, has been reported to exhibit potent and selective antineoplastic effects against cervical cancer cells. In this study, the potential of SF1 to inhibit the stemness of cervical cancer stem-like cells has been evaluated. SF1 extraction was carried out using the dry column vacuum chromatography technique. SiHa cell lines were cultured as spheres in CSC-conditioned medium (cervospheres), and the IC50 of SF1 against cervospheres was determined using the OZBlue Cell Viability Kit. The effects of SF1 on the cervosphere’s stemness markers, including CD49f, CK17, SOX2, OCT4, and NANOG, were assessed using a flow cytometry assay. Self-renewal inhibition and anti-tumorigenesis effects of SF1 in cervospheres were evaluated using a sphere formation assay and a xenograft mouse model. The present study shows that SF1 treatment at an IC50 of 17.07 µg/mL inhibited the proliferation, self-renewal, and tumorigenic capacity of SiHa cervospheres in vitro and in vivo. A decrease in the expressions of CK17, SOX2, CD49f, and OCT4 in cervical CSCs indicated that SF1’s inhibitory effects were also associated with the suppression of stemness markers. These results suggest that SF1 possesses an antitumor effect against cervical CSCs and may be regarded as a promising approach to the development of targeted anticancer agents for cervical cancer.

Cancer Stem Cells from Definition to Detection and Targeted Drugs.

Cancers remain the second leading cause of mortality in the world. Preclinical and clinical studies point an important role of cancer/leukaemia stem cells (CSCs/LSCs) in the colonisation at secondary organ sites upon metastatic spreading, although the precise mechanisms for specific actions are still not fully understood. Reviewing the present knowledge on the crucial role of CSCs/LSCs, their plasticity, and population heterogeneity in treatment failures in cancer patients is timely. Standard chemotherapy, which acts mainly on rapidly dividing cells, is unable to adequately affect CSCs with a low proliferation rate. One of the proposed mechanisms of CSC resistance to anticancer agents is the fact that these cells can easily shift between different phases of the cell cycle in response to typical cell stimuli induced by anticancer drugs. In this work, we reviewed the recent studies on CSC/LSC alterations associated with disease recurrence, and we systematised the functional assays, markers, and novel methods for CSCs screening. This review emphasises CSCs' involvement in cancer progression and metastasis, as well as CSC/LSC targeting by synthetic and natural compounds aiming at their elimination or modulation of stemness properties.

Abstract 1815: Optimization of Lin28 inhibitors: A promising therapeutic approach for the suppression of cancer cell proliferation, stemness properties, and spheroid growth

Abstract Background: The pluripotency factor known as Lin28 plays a crucial role in the regulation of cancer cell stem-like characteristics, hence facilitating the formation of cancer and the evolution of therapy-resistant tumors. The protein exerts its function by utilizing its cold shock domain and zinc knuckle domain (ZKD) to engage with the Let-7 pre-microRNA and impede the process of Let-7 production. The implementation of chemical agents to hinder the interaction between Lin28 and Let-7 has been proven as a therapeutic approach for treating aggressive tumors. Methods: Computer-aided drug design techniques were employed in the development of a novel series of small molecule inhibitors, which are derivatives of our previously identified lead compounds Ln115, Ln15, and Ln7. Fluorescent Polarization Assay (FP), and Electrophoretic Mobility Shift Assay (EMSA) were used to screen the chemicals as Lin28-Let7 interaction disruptors using Lin28A/B ZKD domain. To further screen the chemicals, the Incucyte cell proliferation assay was performed using two Lin28A and Lin28B positive cells, IGROV-1, and DuNE cells, respectively. The inhibition of Lin28 activity was subsequently evaluated by measuring Let-7 micro-RNA and Lin28 protein levels using RT-PCR and Western Blot. Then, the metabolism profile was explored using Microsomal Stability Assay. Incucyte proliferation assay and MTS assay determined the IC50 of lead compound in Lin28 positive cells. The anti-stemness properties of the lead compound were subsequently evaluated through assays for stem gene expression levels and colony formation. To evaluate the potential of the lead compound to further move into animal studies, we tested it on a newly developed 3D Neuroendocrine Prostate Cancer (NEPC) model. Results: In this study, we provide a novel set of derivatives, within which, Ln268 was chosen as the lead compound. Ln268 shows higher metabolic stability with a half-life time of 462 min and clearance rate of 5 ul/min/mg, compared to its father compound at 347 min and 46 ul/min/mg, respectively. Ln268 treatment resulted in a time-dependent inhibition of cell growth with an IC50 of 2.5 uM at 72 hrs. Ln268 reduces mRNA levels of stemness and neurodifferentiation markers such as CHGA, CHG, SYP, SCGN, OCT3/4, NANOG, ID4, FOXC1, ALDH1A2, and FOXO3. In the 3D NEPC model, Ln268 suppresses spheroid growth in a dose- and time-dependent manner. Ln268 achieved 100% spheroid growth inhibition at the concentration of 100 uM, while the current commercially available C1632 only reached 50%. Conclusion: We discovered a novel Ln15 derivative named Ln268, which has a better metabolism profile and increased inhibitory activity to distrust Lin28-Let7 interaction and cancer cell proliferation. Ln268 has the potential to be improved and used as an anti-cancer treatment for a variety of malignancies, such as prostate cancer. Citation Format: Victor M. Matias-Barrios, Mariia Radaeva, Graciella Rosellinny, Artem Cherkasov, Xuesen Dong. Optimization of Lin28 inhibitors: A promising therapeutic approach for the suppression of cancer cell proliferation, stemness properties, and spheroid growth [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 1815.

Abstract 4468: Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver cancer

Abstract Hepatocellular carcinoma (HCC) represents a major global health challenge, characterized by its heterogeneity and aggressive nature. Solid evidence has now suggested that tumor growth is driven by subpopulations of tumor cells with less differentiated cell states, called cancer stem cells (CSCs), which govern therapeutic resistance and tumor recurrence. Therefore, characterization of CSCs and their associated oncogenic signaling pathways is crucial to the development of more specific and sensitive HCC prognostic markers and to the design of novel targeted therapies against CSCs for HCC treatment. In search of the altered genes and pathways that are tightly associated with cancer stemness trait in HCC, we exploited the stemness and oncogenic dedifferentiation signature to mine the HCC dataset of the Cancer Genome Atlas. Phosphoinositide signaling was identified to be enriched and downregulated in the transcriptomes of HCC patients with strong stemness and dedifferentiated signature. Subsequent clinical correlation analysis found inositol polyphosphate-5-phosphatase B (INPP5B) to be the top downregulated gene in phosphoinositide signaling with its expression correlated with poor survival in HCC patients. Functional studies showed that INPP5B overexpression reversed the oncogenic and stemness properties while INPP5B knockout promoted tumor growth in immunocompetent mice. Subsequent RNA sequencing and molecular assays revealed INPP5B deregulation to be associated with lipid metabolism remodeling. Collectively, our results suggest an intricate relationship between phosphoinositide signaling and lipid metabolism through the regulation of INPP5B in HCC. The findings may open new avenue for future research aimed at devising targeted therapies to interfere with liver CSCs and enhance the prognosis of HCC patients. Citation Format: Linglin Liu, Yifei Wang, Yunong Xie, Man Tong. Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver 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 4468.

Abstract 7376: Modeling the dynamics of lung cancer cells along the epithelial−mesenchymal spectrum in the stem and non-stem cell compartments

Abstract Epithelial-mesenchymal transition (EMT) is a crucial biological process during development which also appears to play a fundamental role in the dissemination of cancer cells. It is known that hybrid states (H), in which cells have mixed epithelial (E) and mesenchymal (M) properties, are particularly conductive to metastasis but the possibility of seeding secondary lesions is also closely linked to the intrinsic tumor initiation potential (stemness). An overall vision taking into account both the EMT spectrum and stemness properties could therefore be the key to fully understanding the metastatic process. In this study we aim to build a mathematical model capable of describing the transitions in the EMT spectrum distinguishing stem from non-stem cell subpopulations in lung cancer cells. Using E-cadherin and N-cadherin staining we evaluated experimentally by flow-cytometry the modulation of epithelial (E), hybrid (H) and mesenchymal (M) phenotypes in A549 lung cancer cells after the induction of EMT by TGFβ treatment and subsequent return to steady state after TGFβ withdrawal. Stem and non-stem populations were identified with the previously validated CD133 marker. To mathematically describe the cellular dynamics we used a system of differential equations assuming transitions E-H, E-M and H-M, for stem and non-stem compartments initially treated as decoupled. Parameters used in the equations were the three switching rates corresponding to the three transitions allowed. To test the robustness of the model we solved the equation multiple times (500) by first stochastically changing the initial conditions and then the initial parameters, obtaining the respective parameters optimized by minimizing the residual between experimental data and model. To decouple the parameters, equations were then solved stochastically 1000 times changing two of three initial parameters, obtaining the optimized third parameter and the respective minimal residual. Parameters fitting closely the experimental data during TGFβ-mediated EMT induction were obtained, indicating that the transition from H to M appears to be absent in the non-stem population but present in the stem population, while the transition from E to M is always present. We also observed that after TGFβ withdrawal the inclusion of an epigenetic decay factor is necessary to model the latency period before MET insurgence when EMT is previously induced in the system. We can conclude that during EMTinduction, the dynamics in the non-stem compartment can be described by a bifurcation model in which population E becomes either M or H and remains permanently in that state, stressing the stability of the H state and also that E cells might have a predisposition to transform into M or H. Furthermore, a portion of H stem cells is able transform into M during EMT and therefore appears more plastic which could underscore a greater metastatic potential. Citation Format: Arianna Di Bernardo, Sarthak Sahoo, Melissa Balsamo, Elsa Quaranta, Mohit Kumar Jolly, Giulia Bertolini, Luca Roz. Modeling the dynamics of lung cancer cells along the epithelial−mesenchymal spectrum in the stem and non-stem cell compartments [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 7376.

Abstract 7077: The oncogenic transcription factor ERG has distinct roles and signaling in different prostate cancer cell types

Abstract ERG is the most common oncogene in prostate cancer, as it is aberrantly expressed in about 50% of cases due to the TMPRSS2/ERG gene rearrangement. ERG is not normally expressed in the adult prostate. The normal function of ERG is confined to hematopoietic stem cells and blood vessels. Aberrant expression of ERG in prostate epithelial cells is oncogenic when combined with mutations that activate the PI3K/AKT signaling pathway. The RAS/MAPK pathway can also regulate ERG function through direct phosphorylation of ERG by ERK. The function of these regulatory events and how they can be exploited to develop therapy for ERG-positive prostate cancer remains unclear. Here we find that ERG expression in prostate epithelial cells with low PI3K/AKT pathway activity drives epithelial to mesenchymal transition and stemness properties. This function requires ERG phosphorylation via a positive feedback loop with the TLR4 signaling pathway, and can be repressed by a TLR4 inhibitor. However, activation of the PI3K/AKT pathway rearranges the ERG cistrome and alters ERG function, where ERG now promotes luminal epithelial differentiation. In the presence of activated AKT, new ERG targets include genes involved in vascular development and ERG phosphorylation is regulated through a positive feedback loop with the VEGF signaling pathway. Despite these differences in cellular differentiation, we find that ERG can promote stemness and tumorigenesis in both AKT-high and AKT-low cellular backgrounds. ERG functions in prostate epithelial cells appear to mirror ERG functions in hematopoietic stem cells and blood vessels. These findings suggest strategies of combinatorial inhibition of ERG function through signaling pathway inhibition. Citation Format: Saranya Rajendran, Peter C. Hollenhorst. The oncogenic transcription factor ERG has distinct roles and signaling in different prostate cancer cell types [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 7077.

Abstract 5433: Functional involvement of ribosomal protein L9 to colorectal cancer stemness

Abstract We have previously demonstrated that targeting of ribosomal protein L9 (RPL9) suppresses the growth of colorectal cancer (CRC) via the inactivation of Id-1/NF-κB signaling axis which is known to augment tumor stemness. Thus we aimed in this study to investigate whether the function of RPL9 was associated with CRC stemness properties. It was initially evidenced that inhibition of RPL9 expression reduces the migration and invasion abilities of HT29 parental cell population. We have then sorted out CD133+ cancer stem cells (CSCs) from HT-29 parental cell culture and treated RPL9-specific siRNAs to the isolated CSCs to observe the effect of RPL9 targeting on stemness. As results, knockdown of RPL9 significantly suppressed the proliferation potential and sphere forming capacity of CD133+ HT29 CSCs accompanying with the reduction in CD133 and Id-1 levels. Reflecting these molecular alterations, targeting RPL9 also inhibited the abilities of migration and invasion in CD133+ HT29 CSCs population. Taken together, these findings suggest that RPL9 could be a therapeutic target for both primary CRC treatment and the prevention of metastasis and/or recurrence. Citation Format: Eun-Hye Jeon, Keun Uk Park, Hun-Mo Ryoo, Ilseon Hwang, Yun-Han Lee. Functional involvement of ribosomal protein L9 to colorectal cancer stemness [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 5433.

Abstract 5451: FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in breast cancer

Abstract Cancer stem cells (CSCs) pose a significant clinical challenge due to their inherent resistance to therapies and prevalence in cancer recurrence. CSCs have the ability for self-renewal and to adapt to different microenvironments during dissemination and seeding into distal organs, and these properties are critical in metastasis. We and others have shown that epithelial to mesenchymal transition (EMT) drives the acquisition of stem cell properties, particularly via the forkhead box protein C2 (FOXC2) transcription factor. FOXC2 is a master regulator of EMT and is necessary for the induction and maintenance of CSC properties. Importantly, FOXC2 is not expressed in most adult tissues except adipocytes, in which it dictates several aspects of lipid metabolism. Additionally, metabolic changes during EMT can equip cancer cells with enhanced survival mechanisms during tumor progression and metastasis. We hypothesized that FOXC2 regulates metabolism in EMT-induced cells, facilitating stem cell properties and metastatic competence. To determine the downstream metabolic genes regulated by FOXC2 and that enhance stemness properties in cancer cells, we performed gene expression profiling and identified fatty acid binding protein 4 (FABP4), which is known to be involved in lipid metabolism, especially in adipocytes and macrophages. We demonstrate that inhibition of FOXC2 expression using shRNA in EMT-induced cells markedly reduces FABP4 expression. In agreement with this, we found that the overexpression of FOXC2 expression rescues the expression of FABP4. In addition, FOXC2 binds to the FABP4 promoter and regulates its expression suggesting that FABP4 is a direct target of FOXC2. We demonstrate that both lipid uptake and CSC formation is inhibited in a dose-dependent manner using a FABP4-specific small molecule inhibitor. Additional experiments are underway to assess the role of FABP4 in CSC formation in vivo and in contributions to metastasis, using our small molecule inhibitor in mouse models of metastatic breast cancer. Overall, our data indicate that FABP4 is a key regulator of stemness in breast cancer cells and targeting the FOXC2—FABP4 axis may provide therapeutic benefits for breast cancer. This work also provides insights into the metabolic shifts that occur during EMT and CSC formation, which warrants further investigation into these mechanisms. Citation Format: Breanna R. Demestichas, Vinita Shivakumar, Esmeralda Ramirez-Peña, Joseph Taube, Petra den Hollander, Sendurai A. Mani. FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in 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 5451.

N6-methyladenosine reader hnRNPA2B1 recognizes and stabilizes NEAT1 to confer chemoresistance in gastric cancer.

Chemoresistance is a major cause of treatment failure in gastric cancer (GC). Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is an N6-methyladenosine (m6A)-binding protein involved in a variety of cancers. However, whether m6A modification and hnRNPA2B1 play a role in GC chemoresistance is largely unknown. In this study, we aimed to investigate the role of hnRNPA2B1 and the downstream mechanism in GC chemoresistance. The expression of hnRNPA2B1 among public datasets were analyzed and validated by quantitative PCR (qPCR), Western blotting, immunofluorescence, and immunohistochemical staining. The biological functions of hnRNPA2B1 in GC chemoresistance were investigated both in vitro and in vivo. RNA sequencing, methylated RNA immunoprecipitation, RNA immunoprecipitation, and RNA stability assay were performed to assess the association between hnRNPA2B1 and the binding RNA. The role of hnRNPA2B1 in maintenance of GC stemness was evaluated by bioinformatic analysis, qPCR, Western blotting, immunofluorescence, and sphere formation assays. The expression patterns of hnRNPA2B1 and downstream regulators in GC specimens from patients who received adjuvant chemotherapy were analyzed by RNAscope and multiplex immunohistochemistry. Elevated expression of hnRNPA2B1 was found in GC cells and tissues, especially in multidrug-resistant (MDR) GC cell lines. The expression of hnRNPA2B1 was associated with poor outcomes of GC patients, especially in those who received 5-fluorouracil treatment. Silencing hnRNPA2B1 effectively sensitized GC cells to chemotherapy by inhibiting cell proliferation and inducing apoptosis both in vitro and in vivo. Mechanically, hnRNPA2B1 interacted with and stabilized long noncoding RNA NEAT1 in an m6A-dependent manner. Furthermore, hnRNPA2B1 and NEAT1 worked together to enhance the stemness properties of GC cells via Wnt/β-catenin signaling pathway. In clinical specimens from GC patients subjected to chemotherapy, the expression levels of hnRNPA2B1, NEAT1, CD133, and CD44 were markedly elevated in non-responders compared with responders. Our findings indicated that hnRNPA2B1 interacts with and stabilizes lncRNA NEAT1, which contribute to the maintenance of stemness property via Wnt/β-catenin pathway and exacerbate chemoresistance in GC.

Targeting Galectin 3 illuminates its contributions to the pathology of uterine serous carcinoma

BackgroundUterine serous cancer (USC) comprises around 10% of all uterine cancers. However, USC accounts for approximately 40% of uterine cancer deaths, which is attributed to tumor aggressiveness and limited effective treatment. Galectin 3 (Gal3) has been implicated in promoting aggressive features in some malignancies. However, Gal3’s role in promoting USC pathology is lacking.MethodsWe explored the relationship between LGALS3 levels and prognosis in USC patients using TCGA database, and examined the association between Gal3 levels in primary USC tumors and clinical-pathological features. CRISPR/Cas9-mediated Gal3-knockout (KO) and GB1107, inhibitor of Gal3, were employed to evaluate Gal3’s impact on cell function.ResultsTCGA analysis revealed a worse prognosis for USC patients with high LGALS3. Patients with no-to-low Gal3 expression in primary tumors exhibited reduced clinical-pathological tumor progression. Gal3-KO and GB1107 reduced cell proliferation, stemness, adhesion, migration, and or invasion properties of USC lines. Furthermore, Gal3-positive conditioned media (CM) stimulated vascular tubal formation and branching and transition of fibroblast to cancer-associated fibroblast compared to Gal3-negative CM. Xenograft models emphasized the significance of Gal3 loss with fewer and smaller tumors compared to controls. Moreover, GB1107 impeded the growth of USC patient-derived organoids.ConclusionThese findings suggest inhibiting Gal3 may benefit USC patients.

Improving hematopoietic differentiation from human induced pluripotent stem cells by the modulation of Hippo signaling with a diarylheptanoid derivative

BackgroundThe diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown.MethodWe utilized a defined cocktail of cytokines without serum or feeder followed by the supplementation of ASPP 049 to produce hematopoietic stem/progenitor cells (HSPCs). Flow cytometry and trypan blue exclusion analysis were used to identify nonadherent and adherent cells. Nonadherent cells were harvested to investigate the effect of ASPP 049 on multipotency using LTC-IC and CFU assays. Subsequently, the mechanism of action was explored through transcriptomic profiles, which were validated by qRT-PCR, immunoblotting, and immunofluorescence analysis.ResultThe supplementation of ASPP 049 increased the number of phenotypically defined primitive HSPCs (CD34+CD45+CD90+) two-fold relative to seeded hiPSC colonies, indicating enhanced HSC derivation from hiPSCs. Under ASPP 049-supplemented conditions, we observed elevated HSPC niches, including CD144+CD73− hemogenic- and CD144+CD73+ vascular-endothelial progenitors, during HSC differentiation. Moreover, harvested ASPP 049-treated cells exhibited improved self-renewal and a significantly larger proportion of different blood cell colonies with unbiased lineages, indicating enhanced HSC stemness properties. Transcriptomics and KEGG analysis of sorted CD34+CD45+ cells-related mRNA profiles revealed that the Hippo signaling pathway is the most significant in responding to WWTR1/TAZ, which correlates with the validation of the protein expression. Interestingly, ASPP 049-supplemented HSPCs upregulated 11 genes similarly to umbilical cord blood-derived HSPCs.ConclusionThese findings suggest that ASPP 049 can improve HSC-generating protocols with proliferative potentials, self-renewal ability, unbiased differentiation, and a definable mechanism of action for the clinical perspective of hematopoietic regenerative medicine.Graphical abstract