Stem Cell Renewal Research Articles

Female-germline specific protein Sakura interacts with Otu and is crucial for germline stem cell renewal and differentiation and oogenesis.

During oogenesis, self-renewal and differentiation of germline stem cells (GSCs) must be tightly regulated. The Drosophila female germline serves as an excellent model for studying these regulatory mechanisms. Here, we report that a previously uncharacterized gene CG14545 , which we named sakura , is essential for oogenesis and female fertility in Drosophila . Sakura is predominantly expressed in the ovaries, particularly in the germline cells, including GSCs. sakura null mutant female flies display rudimentary ovaries with germline-less and tumorous phenotypes, fail to produce eggs, and are completely sterile. The germline-specific depletion of sakura impairs Dpp/BMP signaling, leading to aberrant bag-of-marbles ( bam ) expression, resulting in faulty differentiation and loss of GSCs. Additionally, sakura is necessary for normal piwi-interacting RNAs (piRNAs) levels and for proper localization of Ool8 RNA-binding protein (Orb) in developing oocytes. We identified Ovarian Tumor (Otu) as protein binding partner of Sakura, and we found that loss of otu phenocopies loss of sakura in ovaries. Thus, we identified Sakura as a crucial factor for GSC renewal and differentiation and oogenesis, and propose that Sakura and Otu function together in these processes.

SLC1A3 is a novel prognostic biomarker associated with immunity and EMT in hepatocellular carcinoma

PurposeSolute carrier family 1 member 3(SLC1A3), a member of the glutamate transporter family, is implicated in the progression of gastric carcinoma and the renewal of thyroid carcinoma stem cells. The purpose of this work is to use experimental validation and bioinformatics analysis to look at the possible involvement of SLC1A3 in hepatocellular carcinoma (HCC).Materials and methodsWe examined the levels of SLC1A3 within HCC and its implications on immunological and epithelial–mesenchymal transition (EMT) features using the TCGA, ImmPort, and Molecular Signatures databases. The relationship between drug sensitivity and SLC1A3 expression was investigated using the GDSC database. Real-time quantitative polymerase chain reaction (qRT-PCR), Western blotting (WB), and cellular function assays were performed to assess SLC1A3 expression and its carcinogenic effects in HCC.ResultsAccording to our research, SLC1A3 overexpression in HCC is associated with a poor prognosis. Elevated levels of SLC1A3 promote HCC cell motility and invasion and can affect the prognosis of HCC by modifying immune responses and epithelial–mesenchymal transition. SLC1A3 has emerged as a novel prognostic marker in HCC and is associated with resistance to certain antitumor drugs.ConclusionSLC1A3 functions as a cancer-promoting factor contributing to poor HCC prognosis by affecting immune cell infiltration and regulating the EMT process. Elevated SLC1A3 expression may also serve as a predictor of treatment response to specific antitumor drugs.

CSIG-18. YAP/TAZ TARGET GENES MAY DETERMINE THE SELF-RENEWAL AND SURVIVAL OF GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma, a neoplasm arising from neuro-glial stem/progenitor cells, is the most aggressive and lethal form of brain cancer, with a median survival of 14 months. Despite intensive treatments like surgery, radiation, and chemotherapy, recurrence is inevitable, underscoring the urgent need for new treatment strategies. Progress is limited by an incomplete understanding of the biological mechanisms that drive glioblastoma formation and progression, hindering the development of effective therapies. Key molecular pathways, particularly the receptor tyrosine kinases, such as EGFR, and their effector pathways play a critical role in the initiation and maintenance of glioblastoma. Our lab’s research using patient-derived glioblastoma neurospheres and mouse models has shown that the transcriptional co-activators YAP and TAZ are overexpressed downstream of EGFR activity in EGFR-amplified or mutant glioblastoma tumors. YAP/TAZ proteins interact with the TEAD family DNA-binding proteins to promote expression of transcriptional target genes, including SOX2, C-MYC, and EGFR itself in glioblastoma, creating a positive feedback loop that enhances tumor stem cell renewal, proliferation, and survival. Furthermore, our research has demonstrated that inhibiting YAP/TAZ activity with the drug Verteporfin induces apoptosis and promotes growth arrest in patient-derived EGFR-amplified/mutant glioblastoma models. Our findings highlight YAP/TAZ-TEAD as key mediators of glioblastoma stem cell (GSC) self-renewal and survival. Ongoing experiments aim to identify tumor cell-specific YAP/TAZ-TEAD transcriptional target genes predictive of response to YAP/TAZ inhibition in GSCs within EGFR-amplified glioblastomas. Our research seeks to uncover the epigenetic basis of YAP/TAZ dependencies and to determine which genes and cellular processes are critical for tumor stem cell survival, tumor progression, and sensitivity to YAP/TAZ inhibition. These target genes could serve as biomarkers for predicting therapeutic response to YAP/TAZ inhibitors in glioblastoma.

Hedgehog Signalling Pathway and Its Role in Shaping the Architecture of Intestinal Epithelium.

The hedgehog (Hh) signalling pathway plays a key role in both embryonic and postnatal development of the intestine and is responsible for gut homeostasis. It regulates stem cell renewal, formation of the villous-crypt axis, differentiation of goblet and Paneth cells, the cell cycle, apoptosis, development of gut innervation, and lipid metabolism. Ligands of the Hh pathway, i.e., Indian hedgehog (Ihh) and Sonic hedgehog (Shh), are expressed by superficial enterocytes but act in the mesenchyme, where they are bound by a Patched receptor localised on myofibroblasts and smooth muscle cells. This activates a cascade leading to the transcription of target genes, including those encoding G1/S-specific cyclin-D2 and -E1, B-cell lymphoma 2, fibroblast growth factor 4, and bone morphogenetic protein 4. The Hh pathway is tightly connected to Wnt signalling. Ihh is the major ligand in the Hh pathway. Its activation inhibits proliferation, while its blocking induces hyperproliferation and triggers a wound-healing response. Thus, Ihh is a negative feedback regulator of cell proliferation. There are data indicating that diet composition may affect the expression of the Hh pathway genes and proteins, which in turn, induces changes in mucosal architecture. This was shown for fat, vitamin A, haem, berberine, and ovotransferrin. The Hh signalling is also affected by the intestinal microbiota, which affects the intestinal barrier integrity. This review highlights the critical importance of the Hh pathway in shaping the intestinal mucosa and summarises the results obtained so far in research on the effect of dietary constituents on the activity of this pathway.

Notch signaling in digestive system cancers: Roles and therapeutic prospects

Digestive system cancers rank among the most prevalent malignant tumors, maintaining persistently high incidence and mortality rates. Notch signaling activity, often aberrant in esophageal, gastric, hepatic, pancreatic, and colorectal cancers, plays a pivotal role in the initiation, progression, and therapy resistance of these malignancies. As a highly conserved pathway, Notch signaling is integral to cell differentiation, survival, proliferation, stem cell renewal, development, and morphogenesis. Its dysregulation has been increasingly linked to various diseases, particularly digestive system cancers. In these malignancies, altered Notch signaling influences multiple biological processes, including cell proliferation, invasion, cell cycle progression, immune evasion, drug resistance, and stemness maintenance. Understanding the mechanisms of Notch signaling in digestive system cancers is essential for the development of novel targeted therapies. Numerous Notch pathway-targeting drugs are currently in preclinical studies, demonstrating promising efficacy both as monotherapies and in combination with conventional anti-cancer treatments. This review summarizes recent high-quality findings on the involvement of Notch signaling in digestive system cancers, focusing on the expression changes and pathological mechanisms of its dysregulated components. Special emphasis is placed on the potential of translating Notch-targeted approaches into therapeutic strategies, which hold promise for overcoming the limitations of existing treatments and improving the poor prognosis associated with these cancers.

Exploring miRNA therapies and gut microbiome-enhanced CAR-T cells: advancing frontiers in glioblastoma stem cell targeting.

Glioblastoma multiforme (GBM) presents a formidable challenge in oncology due to its aggressive nature and resistance to conventional treatments. Recent advancements propose a novel therapeutic strategy combining microRNA-based therapies, chimeric antigen receptor-T (CAR-T) cells, and gut microbiome modulation to target GBM stem cells and transform cancer treatment. MicroRNA therapies show promise in regulating key signalling pathways implicated in GBM progression, offering the potential to disrupt GBM stem cell renewal. CAR-T cell therapy, initially successful in blood cancers, is being adapted to target GBM by genetically engineering T cells to recognise and eliminate GBM stem cell-specific antigens. Despite early successes, challenges like the immunosuppressive tumour microenvironment persist. Additionally, recent research has uncovered a link between the gut microbiome and GBM, suggesting that gut dysbiosis can influence systemic inflammation and immune responses. Novel strategies to modulate the gut microbiome are emerging, enhancing the efficacy of microRNA therapies and CAR-T cell treatments. This combined approach highlights the synergistic potential of these innovative therapies in GBM treatment, aiming to eradicate primary tumours and prevent recurrence, thereby improving patient prognosis and quality of life. Ongoing research and clinical trials are crucial to fully exploit this promising frontier in GBM therapy, offering hope to patients grappling with this devastating disease.

Poly (A) binding protein 2 is critical for stem cell differentiation during regeneration in the planarian Schmidtea mediterranea.

Post-transcriptional regulation has emerged as a key mechanism for regulating stem cell renewal and differentiation, which is essential for understanding tissue regeneration and homeostasis. Poly(A)-binding proteins are a family of RNA-binding proteins that play a vital role in post-transcriptional regulation by controlling mRNA stability and protein synthesis. The involvement of poly(A) binding proteins in a wide range of cellular functions is increasingly being investigated. In this study, we used the regenerative model planarian organism Schmidtea mediterranea to demonstrate the critical role of poly(A)-binding protein 2 (PABP2) in regulating neoblast maintenance and differentiation. A deficit in PABP2 blocks the transition of neoblasts toward immediate early progenitors, leading to an enhanced pool of non-committed neoblasts and a decreased progenitor population. This is reflected in variations in the transcriptome profile, providing evidence of downregulation in multiple lineages. Thus, an insufficiency of PABP2 resulted in defective formation and organization of tissue, leading to abnormal regeneration. Our study reveals the essential role of PABP2 in regulating genes that mediate stem cell commitment to early progenitors during tissue regeneration.

Structural basis of frizzled 7 activation and allosteric regulation

Frizzleds (ten paralogs: FZD1-10) belong to the class F of G protein-coupled receptors (GPCRs), which remains poorly understood despite its crucial role in multiple key biological functions including embryonic development, stem cell regulation, and homeostasis in the adult. FZD7, one of the most studied members of the family, is more specifically involved in the migration of mesendoderm cells during the development and renewal of intestinal stem cells in adults. Moreover, FZD7 has been highlighted for its involvement in tumor development predominantly in the gastrointestinal tract. This study reports the structure of inactive FZD7, without any stabilizing mutations, determined by cryo-electron microscopy (cryo-EM) at 1.9 Å resolution. We characterize a fluctuating water pocket in the core of the receptor important for FZD7 dynamics. Molecular dynamics simulations are used to investigate the temporal distribution of those water molecules and their importance for potential conformational changes in FZD7. Moreover, we identify lipids interacting with the receptor core and a conserved cholesterol-binding site, which displays a key role in FZD7 association with a transducer protein, Disheveled (DVL), and initiation of downstream signaling and signalosome formation.

Engineered Biomaterials and Model Systems to Study YAP/TAZ in Cancer.

The transcriptional coactivators yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are master regulators involved in a multitude of cancer types and a wide range of tumorigenic events, including cancer stem cell renewal, invasion, metastasis, tumor precursor emergence, and drug resistance. YAP/TAZ are known to be regulated by several external cues and stimuli, such as extracellular matrix stiffness, cell spreading, cell geometry, and shear stress. Therefore, there is a need in the field of cancer research to develop and design relevant in vitro models that can accurately reflect the complex biochemical and biophysical cues of the tumor microenvironment central to the YAP/TAZ signaling nexus. While much progress has been made, this remains a major roadblock to advancing research in this field. In this review, we highlight the current engineered biomaterials and in vitro model systems that can be used to advance our understanding of how YAP/TAZ shapes several aspects of cancer. We begin by discussing current 2D and 3D hydrogel systems that model the YAP/TAZ response to ECM stiffness. We then examine the current trends in organoid culture systems and the use of microfluidics to model the effects of cellular density and shear stress on YAP/TAZ. Finally, we analyze the ongoing pitfalls of the present models used and important future directions in engineering systems that will advance our current knowledge of YAP/TAZ in cancer.

KAT2A and KAT2B prevent double-stranded RNA accumulation and interferon signaling to maintain intestinal stem cell renewal.

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and sequencing of immunoprecipitated double-stranded RNA were used to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions and maintaining intestinal health.

Drosophila: a Tale of regeneration with MYC.

Regeneration is vital for many organisms, enabling them to repair injuries and adapt to environmental changes. The mechanisms underlying regeneration are complex and involve coordinated events at the cellular and molecular levels. Moreover, while some species exhibit remarkable regenerative capabilities, others, like mammals, have limited regenerative potential. Central to this process is the regulation of gene expression, and among the numerous genes involved, MYC emerges as a regulator of relevant processes during regeneration with roles conserved in several species, including Drosophila. This mini-review aims to provide valuable insights into the regeneration process in flies, focusing on significant organs where the role of MYC has been identified: from the imaginal discs, where MYC regulates cell growth, structure, and proliferation, to the gut, where it maintains the balance between renewal and differentiation of stem cells, and the central nervous system, where it influences the activities of neural stem cells and the interaction between glia and neuronal cells. By emphasizing the molecular mechanisms regulated by MYC, its significance in controlling regeneration mechanisms, and its conserved role in flies, we aim to offer valuable insights into the utility of Drosophila as a model for studying regeneration. Moreover, unraveling MYC's function in Drosophila during regeneration may help translate findings into the mechanisms underlying human tissue repair.

Deficiency of ValRS-m Causes Male Infertility in Drosophila melanogaster.

Drosophila spermatogenesis involves the renewal of germline stem cells, meiosis of spermatocytes, and morphological transformation of spermatids into mature sperm. We previously demonstrated that Ocnus (ocn) plays an essential role in spermatogenesis. The ValRS-m (Valyl-tRNA synthetase, mitochondrial) gene was down-regulated in ocn RNAi testes. Here, we found that ValRS-m-knockdown induced complete sterility in male flies. The depletion of ValRS-m blocked mitochondrial behavior and ATP synthesis, thus inhibiting the transition from spermatogonia to spermatocytes, and eventually, inducing the accumulation of spermatogonia during spermatogenesis. To understand the intrinsic reason for this, we further conducted transcriptome-sequencing analysis for control and ValRS-m-knockdown testes. The differentially expressed genes (DEGs) between these two groups were selected with a fold change of ≥2 or ≤1/2. Compared with the control group, 4725 genes were down-regulated (dDEGs) and 2985 genes were up-regulated (uDEGs) in the ValRS-m RNAi group. The dDEGs were mainly concentrated in the glycolytic pathway and pyruvate metabolic pathway, and the uDEGs were primarily related to ribosomal biogenesis. A total of 28 DEGs associated with mitochondria and 6 meiosis-related genes were verified to be suppressed when ValRS-m was deficient. Overall, these results suggest that ValRS-m plays a wide and vital role in mitochondrial behavior and spermatogonia differentiation in Drosophila.

UM171 suppresses breast cancer progression by inducing KLF2

PurposeBreast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action.MethodsThe inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells.ResultsUM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21CIP1. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture.ConclusionThese results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.

Chemokine CXCL8 and its receptors as markers of colorectal cancer

BACKGROUND: The chemokine CXCL8 and its receptor are involved in the activation and transport of inflammatory mediators and regulate the proliferation and renewal of cancer stem cells in colon cancer. It is believed that CXC signaling may be associated with poor prognosis in colorectal cancer. AIM: To study the possibilities of using CXCL8-CXCR1/2 pathway indicators as markers in colorectal cancer. MATERIAL AND METHODS: Isolation of ribonucleic acid (RNA) from histological sections of tumors obtained intraoperatively from 59 patients diagnosed with colorectal cancer was carried out using magnetic particles, and quantitative polymerase chain reaction in real time was performed. Calculation of the normalized expression of the CXCL8 and CXCR1 genes relative to the referee gene was done using special software. Statistical data processing was performed using Statistica 3.0, BioStat v. 7.1 programs. Comparisons of characteristics were performed using the Mann–Whitney U test. Cox and Kaplan–Meier criteria were used to analyze overall and disease-free survival. RESULTS: The expression of CXCL8 in intestinal adenocarcinoma cells with low differentiation [Me (Q1–Q3) — 8.770 (1.127–1.114)] was significantly higher than in the groups of moderately and well differentiated tumors (p=0.004 and p=0.012, respectively); and in tumor tissue refractory to chemotherapy, it was significantly higher [4.374 (2.052–7.045)] compared to resistant [2.200 (1.388–5.037); p=0.008] and sensitive [1.624 (0.739–2.586); p=0.042]. The level of CXCR1 messenger RNA in tumor tissue was increased in the presence of BRAF mutation [3.645 (0.801–1.090); p=0.009] and low tumor differentiation [6.965 (3.938–12.225); p=0.002], as well as in tumor tissue refractory to FOLFOX/XELOX chemotherapy [46.224 (27.580–83.570); p=0.0009]. CONCLUSION: Expression of components of the CXCL8-CXCR1/2 signaling pathway in tumor tissue may be a marker of sensitivity to FOLFOX/XELOX chemotherapy in colorectal cancer.

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture

BackgroundHyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications.MainHere we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former.ConclusionCultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

The effect of turmeric extract (Curcuma longa) on IL-6 expression

Background: Breast cancer in Indonesia is reported to have reached 68,858 out of 396,914 new cases of cancer, with an incidence rate of 16.6% based on Globocan 2020 data. Complementary therapies are much sought after for psychological and economic reasons and have minimal side effects. Curcumin has anti-carcinogenic properties. IL-6 plays an important role in the growth and metastasis of breast cancer cells, renewal of breast cancer stem cells, and drug resistance in breast cancer stem cells. Methods: A randomized controlled trial with post-test only group design using artificial female Balb/c strain rats (Mus Musculus) with DMBA-induced mammary adenocarcinoma nodules were reported, rats were divided into four groups randomly, namely, Control (K), Treatment 1: turmeric extract dose 100mg/KgBW (P1), Treatment 2: turmeric extract dose 150mg/KgBW (P2), Treatment 3: turmeric extract dose 200mg/KgBW (P3). Variables under study were IL-6 expression. One Way Anova test, followed by the Post-hoc Test, was used to test the difference among and between groups. Results: The mean value of IL-6 expression in groups K (53,59 ± 8,37), P1 (50,66 ± 9,59), P2 (66,93 ± 9,64), P3 (64,58 ± 5,97). The Post-Hoc test showed significant differences in IL-6 expression from group K against P2 (p=0.038), group P1 against P2 (p=0.015), and P3 (p=0.040). Conclusion: Turmeric extract (Curcuma longa) had an effect on IL-6 expression in the control group and the treatment group.

Mechanically-driven stem cell separation in tissues caused by proliferating daughter cells

The homeostasis of epithelial tissue relies on a balance between the self-renewal of stem cell populations, cellular differentiation, and loss. Although this balance needs to be tightly regulated to avoid pathologies, such as tumor growth, the regulatory mechanisms, both cell-intrinsic and collective, which ensure tissue steady-state are still poorly understood. Here, we develop a computational model that incorporates basic assumptions of stem cell renewal into distinct populations and mechanical interactions between cells. We find that the model generates unexpected dynamic features: stem cells repel each other in the bulk tissue and are thus found rather isolated, as in a number of in vivo contexts. By mapping the system onto a gas of passive Brownian particles with effective repulsive interactions, that arise from the generated flows of differentiated cells, we show that we can quantitatively describe such stem cell distribution in tissues. The interaction potential between a pair of stem cells decays exponentially with a characteristic length that spans several cell sizes, corresponding to the volume of cells generated per stem cell division. Our findings may help understanding the dynamics of normal and cancerous epithelial tissues.

Abstract 469: PIWI-interacting RNA 50485 (piRNA-50485) is a novel lung tumor suppressor

Abstract Introduction: It is crucial to identify new driver genes and associated downstream pathways to facilitate the development of targeted therapies to effectively treat late-stage and metastatic lung cancer. One pathway involves the Ras association domain family 1 (RASSF1) gene which encodes multiple isoforms. Our laboratory has discovered that the RASSF1C isoform exhibits functional characteristics of an oncogene. We discovered that RASSF1C modulates the expression of PIWI-(Drosophila)-like 1 (PIWIL1, a stem cell renewal gene), as well as novel PIWI-interacting RNAs (piRNAs), which are regulators of the stem cell phenotype and have been linked to oncogenesis. While studying this pathway, we have identified novel piRNAs that are modulated by RASSF1C in non-small cell lung cancer (NSCLC) cells that appear to act as oncogenes or tumor suppressors. Methods: We conducted a global microarray screen to identify RASSF1C PIWI-interacting RNA (piRNA) gene targets. Several piRNA genes that are up- and down-regulated by RASSF1C were identified and the expression of selected up-regulated and down-regulated target genes were confirmed by RT-PCR. We evaluated the impact of selected piRNAs on lung cancer cell proliferation and migration in vitro. We also assessed the expression of the validated piRNA target genes in lung tumor and matched normal tissues. Host and target genes for specific piRNAs were identified using a piRNA database (piRNAdb.org). Results: Among the down-regulated piRNAs, we found that restoring piR-50485 expression drastically decreased lung cancer cell proliferation and invasion, suggesting that piR-50485 could be a potent inhibitor of lung cancer cell growth and progression. Consistent with this, we found that piR-50485 expression is significantly down-regulated in about 60% of human lung cancers. We also identified that the ZHX3 gene (a transcription repressor), which is down-regulated in human lung cancers, is the host gene for piR-50485. In addition, we identified the ZNF618 gene (a transcription co-regulator) that is up-regulated in lung tumor tissue, is a piR-50485 target gene. Consistent with this, treatment of lung cancer cells with piR-50485 mimics resulted in a significant decrease of ZNF618 protein levels, cell proliferation, and migration. Further, increased expression levels of ZHX3 are associated with higher lung cancer patient survival, while increased ZNF618 levels are associated with lower survival. Our novel findings suggest the hypothesis that the piR-50485-ZHX3-ZNF618 gene axis could negatively impact lung cancer cell growth and progression. Conclusion: Exploring the impact of the piR-50485-ZHX3-ZNF618 gene axis on lung cancer growth is a novel concept about which nothing is currently known. We are studying piR-50485 to determine if it can be an effective therapy for lung cancer metastasis, and whether it provides important prognostic information about lung cancer outcomes. Citation Format: Mark E. Reeves, Yousef G. Amaar. PIWI-interacting RNA 50485 (piRNA-50485) is a novel lung tumor suppressor [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 469.

Frequent Inactivation of Secreted Frizzled-Related Protein 2 during the Development of Cervical Carcinoma: Identification of Susceptible Alleles and Clinical Implications

ABSTRACT Background: In this study, importance of SFRP2, wnt stem cell renewal pathway antagonist, in the development of cervical cancer (CACX) was evaluated Aims and Objectives: Alterations (expression/ methylation/ deletion) of SFRP2 were analysed in primary cervical lesions of different clinical stages followed by their correlation with different clinicopathological parameters. Then, susceptible allele(s) of SFRP2 was identified through case control study followed by and in vitro validation. Results: The mRNA expression of SFRP2 was gradually reduced with progression of CACX. In immunohistochemistry, SFRP2 membrane expression was mainly present in the spinous layers of normal cervical epithelium and its reduced protein expression in CACX samples showed concordance with mRNA expression. Frequent deletion/ methylation of SFRP2 were seen to be associated with development of cervical cancer. Methylation of SFRP2 was prevalently associated with early invasive lesions (stage I/II) while, deletion with late invasive lesions (stage III/IV). Overall alterations (deletion/ methylation) of SFRP2 were significantly increased from premalignant CIN to stage-I/II samples followed by comparable change to the next stage (stage III/IV) samples. Moreover, deletion and/or methylation of SFRP2 were associated with poor prognosis of the patients. In a case control study, out of its seven microsatellite alleles infrequent SFRP_CA15/16 alleles along with frequent SFRP_CA17 allelewere found to be associated with CACX development. Comparatively reduced expression (mRNA/ protein) of SFRP2 was seen in the tumor adjacent normal cervical epithelium having SFRP_CA15/16 alleles than the other alleles. This has been further validated in in vitro luciferase promoter activity assay where SFRP_CA16 repeat showed high reduced activity followed by SFRP_CA15 repeat than the other repeats. Conclusion: Thus, our data showed that presence of the infrequent susceptible alleles along with deletion/methylation might have synergistic effect on frequent inactivation of SFRP2 during development of CACX.

The Role of the Notch Signaling Pathway in the Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells.

Human umbilical cord mesenchymal stem cells (hUCMSCs) exhibit potent self-renewal and multilineage differentiation characteristics. They have garnered substantial attention within the domain of regenerative medicine owing to their therapeutic potential, such as in tissue repair, regeneration, immunomodulation, anti-inflammation, angiogenesis, wound healing, neuroprotection, and neuroregeneration. The process of fate determination is initiated by multiple signaling molecules. During development and tissue homeostasis, the Notch signaling pathway assumes a pivotal function in cell differentiation and the renewal of stem cells. A growing body of research has revealed that the Notch signaling pathwayplays a pivotal role in hUCMSC proliferation and differentiation. The latest progress concerning the crucial functions of the Notch signaling pathway in maintaining homeostasis and determining the cell fate of hUCMSCs is summarized. Furthermore, the authors also summarized the mediators related to the Notch signaling pathway in hUCMSC differentiation, as well as the pathway alterations and mechanisms involved in hUCMSC therapy.