Cancer Stem Cell Renewal Research Articles

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.

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.

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.

CDKN1A/p21 in Breast Cancer: Part of the Problem, or Part of the Solution?

Cyclin-dependent kinase inhibitor 1A (Cip1/Waf1/CDKN1A/p21) is a well-established protein, primarily recognised for its pivotal role in the cell cycle, where it induces cell cycle arrest by inhibiting the activity of cyclin-dependent kinases (CDKs). Over the years, extensive research has shed light on various additional mechanisms involving CDKN1A/p21, implicating it in processes such as apoptosis, DNA damage response (DDR), and the regulation of stem cell fate. Interestingly, p21 can function either as an oncogene or as a tumour suppressor in these contexts. Complicating matters further, the expression of CDKN1A/p21 is elevated in certain tumour types while downregulated in others. In this comprehensive review, we provide an overview of the multifaceted functions of CDKN1A/p21, present clinical data pertaining to cancer patients, and delve into potential strategies for targeting CDKN1A/p21 as a therapeutic approach to cancer. Manipulating CDKN1A/p21 shows great promise for therapy given its involvement in multiple cancer hallmarks, such as sustained cell proliferation, the renewal of cancer stem cells (CSCs), epithelial-mesenchymal transition (EMT), cell migration, and resistance to chemotherapy. Given the dual role of CDKN1A/p21 in these processes, a more in-depth understanding of its specific mechanisms of action and its regulatory network is imperative to establishing successful therapeutic interventions.

Enhancement of TKI sensitivity in lung adenocarcinoma through m6A-dependent translational repression of Wnt signaling by circ-FBXW7

BackgroundTyrosine kinase inhibitors (TKIs) that specifically target mutational points in the EGFR gene have significantly reduced suffering and provided greater relief to patients with lung adenocarcinoma (LUAD). The third-generation EGFR-TKI, Osimertinib, has been successfully employed in clinical treatments to overcome resistance to both original and acquired T790M and L858R mutational points. Nevertheless, the issue of treatment failure response has emerged as an insurmountable problem.MethodsBy employing a combination of multiple and integrated approaches, we successfully identified a distinct population within the tumor group that plays a significant role in carcinogenesis, resistance, and recurrence. Our research suggests that addressing TKI resistance may involve targeting the renewal and repopulation of stem-like cells. To investigate the underlying mechanisms, we conducted RNA Microarray and m6A Epi-Transcriptomic Microarray analyses, followed by assessment of transcription factors. Additionally, we specifically designed a tag to detect the polypeptide circRNA-AA, and its expression was confirmed through m6A regulations.ResultsWe initially identified unique molecular signatures present in cancer stem cells that contributed to poor therapeutic responses. Activation of the alternative Wnt pathway was found to sustain the renewal and resistant status of these cells. Through bioinformatics analysis and array studies, we observed a significant decrease in the expression of circFBXW7 in Osimertinib-resistant cell lines. Notably, the abnormal expression pattern of circFBXW7 determined the cellular response to Osimertinib. Functional investigations revealed that circFBXW7 inhibits the renewal of cancer stem cells and resensitizes both resistant LUAD cells and stem cells to Osimertinib. In terms of the underlying mechanism, we discovered that circFBXW7 can be translated into short polypeptides known as circFBXW7-185AA. These polypeptides interact with β-catenin in an m6A-dependent manner. This interaction leads to reduced stability of β-catenin by inducing subsequent ubiquitination, thereby suppressing the activation of canonical Wnt signaling. Additionally, we predicted that the m6A reader, YTHDF3, shares common binding sites with hsa-Let-7d-5p. Enforced expression of Let-7d post-transcriptionally decreases the levels of YTHDF3. The repression of Let-7d by Wnt signaling releases the stimulation of m6A modification by YTHDF3, promoting the translation of circFBXW7-185AA. This creates a positive feedback loop contributing to the cascade of cancer initiation and promotion.ConclusionsOur bench study, in vivo experiments, and clinical validation have unequivocally shown that circFBXW7 effectively inhibits the abilities of LUAD stem cells and reverses resistance to TKIs by modulating Wnt pathway functions through the action of circFBXW7-185AA on β-catenin ubiquitination and inhibition. The regulatory role of circRNA in Osimertinib treatment has been rarely reported, and our findings reveal that this process operates under the influence of m6A modification. These results highlight the tremendous potential of this approach in enhancing therapeutic strategies and overcoming resistance to multiple TKI treatments.

Wnt/β-catenin signaling pathway inhibitors, glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside-1, diosmin, and rutin in medicinal plants have better binding affinities and anticancer properties: Molecular docking and ADMET study.

Wnt/β-catenin signaling pathway plays a role in cancer development, organogenesis, and embryogenesis. The abnormal activation promotes cancer stem cell renewal, proliferation, and differentiation. In the present study, molecular docking simulation and ADMET studies were carried out on selected bioactive compounds in search of β-catenin protein inhibitors for drug discovery against cancer. Blind docking simulation was performed using PyRx software on Autodock Vina. β-catenin protein (PDB ID: 1jdh) and 313 bioactive compounds (from PubChem database) with selected standard anticancer drugs were used for molecular docking. The ADMET properties of the best-performing compounds were calculated using SwissADME and pkCMS web servers. The results obtained from the molecular docking study showed that glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside-1, diosmin, and rutin had the best binding interactions with β-catenin protein based on their binding affinities. Glycyrrhizic acid and solanine had the same and lowest binding energy of -8.5 kcal/mol. This was followed by polyphyllin I with -8.4 kcal/mol, and crocin, hypericin, and tubeimoside-1 which all had a binding energy of 8.1 kcal/mol. Other top-performing compounds include diosmin and rutin with binding energy of -8.0 kcal/mol. The ADMET study revealed that the following compounds glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside-1, diosmin, rutin, and baicalin all violated Lipinski's rule of 5 which implies poor oral bioavailability. However, based on the binding energy score, it was suggested that these pharmacologically active compounds are potential molecules to be tested against cancer.

Abstract 4955: Kurarinone, a natural notch signaling inhibitor and modulator of breast cancer stemness

Abstract Aberrant activation of Notch signaling pathway plays an important role in breast cancer development, progression, and stem cell renewal. Activation of Notch signaling is triggered by the interaction of Notch receptors with different ligands expressed on neighboring cells that bind and induce a second cleavage by a membrane-tethered metalloprotease. The remaining membrane-bound fragment is cleaved by γ-secretase (GS) releasing the Notch Intracellular Domain (NICD), which translocate to the nucleus to activate the expression of Notch target genes. Current indirect approaches to inhibit Notch signaling are based on agents that bind to GS substrates. In the search of novel Notch signaling inhibitor(s), we screened a number of plant flavonoids for their binding potential to GS substrate. Our in-silico study identified kurarinone (KU), a lavandulyl flavanone, having ability to bind to GS complex. The KU-GS complex was structurally and energetically stable during the molecular dynamics simulation period. Interestingly, dose and time dependent toxicity of KU was observed in T47D and MDA-MB-231 breast cancer cells at 1-25µM concentration. KU treatment of cells led to inhibition in cyclin D1 expression, arrest of cells in the G1 phase of the cell cycle and subsequently undergoes apoptosis. KU-mediated apoptosis was a result of altered mitochondria membrane potential and increased ROS production. In dual-luciferase Notch promoter assay, KU inhibited GS-mediated NICD production in transfected HEK293 cells. Furthermore, KU treatment decreased the mammosphere formation potential of MCF-7 cells through altered gene expression involved in self-renewal and stemness. KU treated MDA-MB-231 cells and MCF-7 derived mammosphere showed downregulation of Notch signaling through alteration in the expression of downstream genes such as Hey1, Hes1, and E-cadherin at both transcript and protein levels. Taken together, the present study identified KU as a novel natural Notch signaling inhibitor that could provide a novel and safer approach for the treatment of breast cancer through reduction in stemness and self-renewal properties. Citation Format: Prem Prakash Kushwaha, Mohd Shuaib, Kumari Sunita Prajapati, Atul Kumar Singh, Mahesh Kulharia, Shashank Kumar, Sanjay Gupta. Kurarinone, a natural notch signaling inhibitor and modulator of breast cancer stemness. [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 4955.

Abstract 1743: miR-1307-5p reverses cisplatin resistance in oral squamous cell carcinomas (OSCC) by targeting EHF, a modulator of stemness genes

Abstract Background: We have previously identified miR-1307-5p is exclusively expressed in OSCC tumors and is significantly upregulated in chemo-resistant patients. In this study, we investigate the mechanism of miR-1307-5p in conferring therapeutic refractoriness and demonstrate reversal of cisplatin induced chemoresistance. This study provides a potentially new therapeutic target for treating recurrent oral carcinomas. Methods: Expression of miR-1307-5p was validated in OSCC patients (n=86), OECM1 cell line and cisplatin resistant CD44+ subpopulation. miR-1307-5p was knocked down in OECM1 and CD44+ cells and its effect on cell proliferation, apoptosis, cell cycle, migration, invasion and sphere forming abilities was measured. We examined the transcriptome of these patients (n=30) and combined this data with The Cancer Genome Atlas (TCGA) dataset for target prediction (TargetScan) and network analysis (Ingenuity Pathway Analysis). The hub genes were considered to be potential targets of miR-1307-5p and were validated by luciferase assay and Western Blotting. Results: Sequencing analysis revealed exclusive expression of miR-1307-5p in the OSCC patient cohort with significant upregulation in chemo-resistant cases (FC:4.82 ± 2.38, p-value:0.01). miR-1307-5p upregulation separated the chemoresistant and remission cohorts (p<0.05). Transfection-mediated silencing of miR-1307-5p in OECM1(FC:0.009 ± 1.5, p:0.01) and CD44+ (FC:0.06 ± 2.3, p:0.01) cells restrained the cell proliferation rates (p<0.01) and revealed a significant sub G0 population of OECM1 (20±5%) and CD44+(18.7±3%) cells, indicating early apoptosis. A significant Annexin V+/PI- population (OECM1: 40%±3.36%; CD44+: 23%±3.36%) confirmed induction of apoptosis, post-silencing. A 2 fold reduction in the sphere-forming ability and invasive potentials of CD44+ cells observed post-knockdown (p>0.05). Combinatorial treatment of cisplatin and miR-1307-5p inhibitor reduced the proliferation rate of CD44+ cells by 66.23% in comparison to standalone treatment of the inhibitor (p:0.034). Thus, indicating role of miR-1307-5p in reversal of chemo-resistance. Gene target identification revealed miR-1307-5p could promote therapeutic refractoriness by suppressing the expression of EHF, which regulates cancer stem cell renewal by targeting Sox-2, Oct-4 and Nanog. Dual-luciferase gene reporter assay revealed direct suppression of EHF by miR-1307-5p. Further, knockdown of miR-1307-5p increased endogenous EHF mRNA and protein expression in cancer cells. Conclusion: miR1307-5p downregulates EHF which is an inhibitor of many stemness genes like Sox-2, Oct-4 and Nanog. Knockdown of miR-1307-5p reverses Cisplatin resistance by upregulating EHF thus suppressing the emergence of cancer stem cells in OSCC. Citation Format: Aditi Patel, Shanaya Patel, Shreya Lotia, Vivek Tanavde. miR-1307-5p reverses cisplatin resistance in oral squamous cell carcinomas (OSCC) by targeting EHF, a modulator of stemness genes [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 1743.

TACH101, a first-in-class pan-inhibitor of KDM4 histone demethylase.

Histone lysine demethylase 4 (KDM4) is an epigenetic regulator that facilitates the transition between transcriptionally silent and active chromatin states by catalyzing the removal of methyl groups on histones H3K9, H3K36, and H1.4K26. KDM4 overamplification or dysregulation has been reported in various cancers and has been shown to drive key processes linked to tumorigenesis, such as replicative immortality, evasion of apoptosis, metastasis, DNA repair deficiency, and genomic instability. KDM4 also plays a role in epigenetic regulation of cancer stem cell renewal and has been linked to more aggressive disease and poorer clinical outcomes. The KDM4 family is composed of four main isoforms (KDM4A-D) that demonstrate functional redundancy and cross-activity; thus, selective inhibition of one isoform appears to be ineffective and pan-inhibition targeting multiple KDM4 isoforms is required. Here, we describe TACH101, a novel, small-molecule pan-inhibitor of KDM4 that selectively targets KDM4A-D with no effect on other KDM families. TACH101 demonstrated potent antiproliferative activity in cancer cell lines and organoid models derived from various histologies, including colorectal, esophageal, gastric, breast, pancreatic, and hematological malignancies. In vivo , potent inhibition of KDM4 led to efficient tumor growth inhibition and regression in several xenograft models. A reduction in the population of tumor-initiating cells was observed following TACH101 treatment. Overall, these observations demonstrate the broad applicability of TACH101 as a potential anticancer agent and support its advancement into clinical trials.

Pyrvinium Pamoate: Past, Present, and Future as an Anti-Cancer Drug.

Pyrvinium, a lipophilic cation belonging to the cyanine dye family, has been used in the clinic as a safe and effective anthelminthic for over 70 years. Its structure, similar to some polyaminopyrimidines and mitochondrial-targeting peptoids, has been linked with mitochondrial localization and targeting. Over the past two decades, increasing evidence has emerged showing pyrvinium to be a strong anti-cancer molecule in various human cancers in vitro and in vivo. This efficacy against cancers has been attributed to diverse mechanisms of action, with the weight of evidence supporting the inhibition of mitochondrial function, the WNT pathway, and cancer stem cell renewal. Despite the overwhelming evidence demonstrating the efficacy of pyrvinium for the treatment of human cancers, pyrvinium has not yet been repurposed for the treatment of cancers. This review provides an in-depth analysis of the history of pyrvinium as a therapeutic, the rationale and data supporting its use as an anticancer agent, and the challenges associated with repurposing pyrvinium as an anti-cancer agent.

CSIG-07. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS IN GLIOBLASTOMA

Abstract P53 is frequently mutated in most human cancers, including glioblastoma (GBM). Many p53 mutants acquire gain-of-function oncogenic effects through only partially understood mechanisms. To investigate the role of gain-of-function mutant p53 (MUT-p53) in GBM, we performed ChIP-seq of wildtype p53 (WT-p53) and MUT-p53 GBM cell lines. Among 2834 unique peaks reads in MUT-p53 cells, we found 242 long non-coding RNAs (lncRNAs) with up to 145 fold enrichment relative to WT-p53. LncRNAs regulate many molecular and cellular functions, including gene expression, cell proliferation, death, cancer stem cell renewal and differentiation. We selected lncRNAs SOX21-AS1 and LINC00643 with highly enriched binding by MUT-p53 and investigated their expressions and functions in the p53 pathway. We performed ChIP confirmation of MUT-p53 binding to the promoters of these lncRNAs. We found that these lncRNAs are deregulated in GBM and correlated with GBM patient survival in the TCGA database. To investigate the functions of these LncRNAs, we knocked down their expressions by siRNA, and found significant cell death induced by si-SOX21-AS1, but not by si-LINC00643. Overexpression of LINC00643 in GBM cells led to inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. LINC00643 mediated the effects of MUT-p53. Co-expression of human LINC00643 and its mouse homologous in a RCAS transgenic mouse model of GBM reduced tumor growth and improved animal survival. To elucidate the mechanisms of action of the lncRNA, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to HIF1a 5’ promoter/enhancer region. Overexpression of LINC00643 in GBM cells at hypoxia growth condition reduced HIF1a mRNA and protein expression. Our study shows for the first time that gain-of-function mutant p53 regulates a subset of lncRNAs and that the lncRNAs mediate the oncogenic effects of the MUT-p53 in GBM.

The Role of MARCKS in Metastasis and Treatment Resistance of Solid Tumors.

Simple SummaryCancer metastasis is a critical event in the progression of solid tumors and is invariably associated with adverse outcomes and mortality. Understanding novel mechanisms or molecules that promote cancer metastasis will facilitate the development of new strategies for cancer treatment. Recently, MARCKS has been studied extensively in several cancers and has been implicated in tumor progression and metastasis. This review summarizes recent advances in the understanding of MARCKS on cancer metastasis, stemness, and therapeutic resistance and provides prospects on targeting MARCKS therapeutically. Specifically, we review the molecular mechanisms and multiple signaling pathways by which MARCKS contributes to the progression and metastasis in solid tumors.The myristoylated alanine-rich C-kinase substrate (MARCKS) is a membrane-associated protein kinase C (PKC) substrate ubiquitously expressed in eukaryotic cells. MARCKS plays important roles in multiple cellular processes, including cell adhesion and motility, mucin secretion, exocytosis, and inflammatory response. Aberrant MARCKS signaling has been observed in the development and progression of multiple cancer types. In addition, MARCKS facilitates cancer metastasis through modulating cancer cell migration and invasion. Moreover, MARCKS contributes to treatment resistance, likely by promoting cancer stem cell renewal as well as immunosuppression. In this review, we describe MARCKS protein structure, cellular localization, and biological functions. We then discuss the role of MARCKS in cancer metastasis as well as its mechanisms of action in solid tumors. Finally, we review recent advances in targeting MARCKS as a new therapeutic strategy in cancer management.

TFEB and TFE3 Depletion Increases Cancer Stem Cell Characteristics and EMT in Triple‐Negative Breast Cancer

Breast cancer is currently the second leading cause of death for women worldwide. One subtype of breast cancer with poor prognosis is triple‐negative breast cancer (TNBC), which is characterized by the lack of expression of estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors. TNBC is difficult to treat due to its drug resistance and aggressiveness. Tumor invasiveness is driven by epithelial‐mesenchymal transition (EMT), which allows cells to acquire mobility to metastasize. EMT is also relevant to maintaining cancer stem cell (CSC) characteristics. Drug resistance is driven by CSCs, which are slow to divide, thus surviving chemotherapies, the standard of treatment. Thus, new treatments that inhibit CSCs and/or EMT in TNBC are highly desirable. TFEB and TFE3, two transcription factors of the MiT‐TFE family that operate as a complex, are involved in regulating lysosome biogenesis and autophagy. Our preliminary data and other studies have shown that TFEB and TFE3 regulate pluripotency and EMT in various in vitro models such as gastric and pancreatic cancer, but this regulation is poorly understood in TNBC. To develop new targeted therapies for TNBC, this study aimed to investigate the effects of depleting TFEB and TFE3 on CSC abundance and EMT. CSC renewal capacity was assessed via mammosphere assays and flow cytometry using CD44 and ALDH1A. The MDA MB 231 TFEBKO cells grew the highest number and largest mammospheres compared to the control, TFE3 knock‐down, and double knock‐out cell lines. TFEBKO and TFEBKOTFE3sh showed increases in CD44 expression and ALDH1A activity. While ALDH1A positivity was 1.16% in controls, TFEBKO and TFEBKOTFE3sh significantly increased the population to 22.6% and 21.9%, respectively. Western blotting and a wound healing assay were used to evaluate EMT marker expression and migration, respectively. Quantification revealed that TFEBKO had a two‐fold increase in protein expression levels of EMT markers Vimentin, Snail, and Slug, while TFE3sh showed higher protein expression levels of Vimentin and Slug. TFEBKOTFE3sh showed higher protein expression levels of Slug. The TFEBKO cell line had the highest rate of migration when compared to the control. In conclusion, the data showed that knockout of TFEB and combined knockout of TFEB and TFE3 resulted in an increase in stem cell character and EMT, which could correlate to increased tumor metastasis and poorer prognosis. These results are promising to investigate how depletion or inhibition of TFEB and TFE3 can affect TNBC tumor cell stem cell character and EMT.

Cananginone Abrogates EMT in Breast Cancer Cells through Hedgehog Signaling.

Cananginones, a family of linear acetogenins found as secondary metabolites in the plant kingdom, show cytotoxicity against several types of cancer cells. We aimed to investigate the efficacy of cananginone and its mechanism as an anti-cancer agent. Our initial screening of Cananginone against HepG2, PC3, A549, and MCF7 cells showed anti-cancer activities and is more potent against MCF7 cells, consistent with the previous report. Next, cell-based assays have revealed that cananginone abrogates cancer stem cell renewal as well as Epithelial-Mesenchymal Transition (EMT) and increased the ROS level beyond the threshold level thus reducing the viability of cancer cells. In the connection of Hh-Gli to EMT, our study indicated that cananginone inhibits Gli1 in a non-canonical pathway. Presumably, this is the first report on the inhibitory activity of cananginone in the Hh pathway and is different from Hh-antagonists cyclopamine and GANT 61 considering the mechanism.

Delineating the Role of Interplay between m6A Machinery Genes and IGF2BP Group of RNA-Binding Proteins in B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Introduction: N-6-methyladenosine (m6A) is the most common, dynamic and reversible RNA modification with implications in various cancers including leukemia. Deregulation of m6A writer METTL3 has been shown to promote disease progression in various cancers, including Acute Myeloid Leukemia(AML). Overexpression of METTL3 led to increase in cell growth and inhibition of apoptosis, thereby promoting leukemia progression. Interestingly, m6A demethylases (erasers) ALKBH5 and FTO have also seen to play a critical role in progression of AML by mediating cancer stem cell renewal. The IGF2BP family of RNA binding, oncofetal proteins have recently been identified as m6A readers and have also been shown to be deregulated in B-ALL. In this work, we have studied the expression of m6A machinery (writers, erasers and readers) in primary (naïve and relapsed) B-ALL patient samples. The percentage of methylated RNA (m6A%) was also evaluated in B-ALL patient samples.Materials and Methods: 91 newly diagnosed (naïve) and 47 relapsed B-ALL pediatric patient bone marrow samples were collected from BRAIRCH, AIIMS, New Delhi. Gene expression of m6A writer (METTL3), readers (IGF2BP1/3) and erasers (ALKBH5, FTO) was studied by RT-qPCR. Peripheral blood (PB) of 20 healthy individuals and 18 uninvolved bone marrow (BM) samples of patients with other malignancies were used as controls. m6A% was also measured in B-ALL patients (naïve n=47, relapsed n=43,) and controls (PB n=20, BM n=16, CD34+ cells from normal donors n=5) by an anti-m6A based colorimetric assay.Results: The ratio of m6A writer METTL3 to m6A eraser ALKBH5 was significantly higher in the naïve and relapsed B-ALL patients as compared to all controls. Interestingly, the ratio of the m6A writer METTL3 to m6A eraser FTO was also significantly high in naïve BM patient sample than controls. The expression of m6A readers IGF2BP1/3 that stabilize the methylated target mRNA, was also studied. IGF2BP1/3 m6A reader was significantly higher in naïve and relapsed patient samples. Increased expression of the writers and readers implied an increase in the m6A levels in B-ALL patients. The m6A% assay showed that the percentage of m6A was significantly higher in naïve and relapsed BM patient samples than both controls corroborating the RT-qPCR data.Discussion: METTL3 m6A methyl transferase has been identified a key factor in mediating the pathogenesis of AML. In our data, we have shown overexpression of METTL3 in B-ALL patient BM samples compared to controls. We have also seen an overexpression of m6A demethylase FTO in B-ALL patient samples. In order to identify the major factor among m6A writers and erasers that might play a role in pathogenesis of B-ALL, we calculated the ratio of m6A writer to m6A eraser. We have observed that ratio of METTL3 to ALKBH5 and METTL3 to FTO was significantly higher in B-ALL patient samples than both the controls. This signifies that overexpression of METTL3 subsequently leading to dysregulated methylation of its targets might influence the development and onset of relapse in B-ALL.It is well known that m6A bound target mRNAs are read by m6A readers like IGF2BPs that stabilize these m6A bound mRNAs leading to overexpression and thereby cancer progression. We have also studied expression of IGF2BP1/3 in B-ALL and seen significant overexpression of both IGF2BP1 and IGF2BP3 in B-ALL samples. These findings indicate a combined dysregulation of m6A writers, erasers and readers in B-ALL. This corroborates with the findings seen in AML, which also shows overexpression of METTL3, ALKBH5 and FTO.Our gene expression studies together point towards an increased percentage of m6A methylated RNA in B-ALL. We have evaluated the percentage of m6A in B-ALL patient samples to confirm our gene expression findings. We observed presence of significantly higher percentage of m6A in B-ALL patient samples (naïve and relapse) than both the controls. m6A% was significantly higher in naïve B-ALL patient samples compared to CD34+ HSCs also.Our findings reveal overall high m6A% in B-ALL, attributed to overexpression of m6A writer METTL3 and m6A readers IGF2BP1/3. This RNA methylation and stabilization might be dysregulated and concentrated in oncogenic genes leading to leukemogenesis. Our results provide a rationale for targeting of these m6A machinery genes dysregulation of which can be instrumental in pathogenesis of B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-34a Alleviates Gemcitabine Resistance in Pancreatic Cancer by Repression of Cancer Stem Cell Renewal.

This study aimed to enhance the sensitivity of pancreatic ductal adenocarcinoma cells by microRNA-34a (miR-34a)-mediated targeting of Notch 1. Cell viability was determined by using an MTT (3-(4,5)-dimethylthiahiazo(-2)-3,5-diphenytetrazoliumromide) assay. The expression levels of miR-34a and relevant mRNAs were determined using quantitative polymerase chain reaction. Protein levels were measured by Western blotting. Cellular stemness was assessed by cell invasiveness and sphere formation assays. A transplanted tumor model was established for in vivo experiments. MicroRNA-34a enhanced gemcitabine sensitivity both in vivo and in vitro. MicroRNA-34a suppressed the stemness and proliferation of pancreatic cancer stem cells. MicroRNA-34a directly associated with Notch 1, which lies upstream of epithelial-mesenchymal transition signaling pathways. MicroRNA-34a sensitized pancreatic cancer cells to gemcitabine treatment by inhibiting Notch 1 signaling in pancreatic cancer stem cells, indicating that miR-34a has the potential to be developed as a novel therapeutic agent for the treatment of gemcitabine-resistant pancreatic ductal adenocarcinoma cells.

Abstract 2583: Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo

Abstract Head and neck cancer (HNC) is the seventh most prevalent cancer worldwide. The five-year survival rate is less than fifty percent despite significant advances in therapeutic approaches. Early diagnosis and treatment can help in prevention and disease management. Studies have reported that dietary intake of Allium vegetables or processed garlic that contains diallyl trisulfide (DATS) lowered the risk of various cancers. However, it has not been investigated for its efficacy and molecular mechanisms against HNC. Herein, we investigated the effect of DATS on HNC UMSCC-22A, UMSCC-22B, and Cal33 cells in vitro. DATS treatment significantly reduced the cell viability of HNC cells. DATS induced significant G2/M phase cell cycle arrest, which resulted in the accumulation of cyclin B1 and induced levels of p21. DATS-induced M phase arrest was attenuated by N-acetyl cysteine (NAC) treatment, which suggested that DATS mediates growth-suppressive effects by reactive oxygen species (ROS) generation. DATS treatment induced mitochondrial dysfunction. DATS induced ROS production led to apoptosis, which was attenuated by NAC treatment. DATS increased proapoptotic protein cleaved caspase-3, cleaved PARP, and Bax/Bcl2 ratio and decreased the levels of antiapoptotic protein XIAP. DATS induced ROS mediated DNA damage which was attenuated by NAC treatment. Further, DATS mediated increase in level of cleaved PARP, accumulation of Cyclin B1 and decrease in XIAP was attenuated by NAC treatment. DATS (1-10 μM) treatment resulted in inhibition of self renewal of HNC cancer stem cells (HNC CSCs) sphere formation. DATS reduced aldehyde dehydrogenase 1 (ALDH1) activity and CD133high/CD44high HNC CSC fraction. DATS-treated SCID mice tumor xenograft also revealed it's in vivo efficacy on HNC CSCs. Further, DATS treatment reduced the tumor weight, volume and reduced the levels of Ki67 cell proliferation marker in UMSCC22B head and neck cancer CD1 nude mice tumor xenograft. DATS treatment decreased tumor incidence and inhibited tumor promotion and progression in twenty two week C57BL6 4-Nitroquinoline 1-oxide (4-NQO)-induced mouse oral carcinogenesis model. DATS treatment reduced the cell proliferation marker Ki67 and increased TUNEL positive apoptotic cells in treatment groups compared to control group. Collectively, DATS inhibited cell proliferation, induced cell cycle arrest, and cell death via apoptosis involving DNA damage, mitochondrial dysfunction, and ROS generation. DATS treatment also reduced the HNC CSC fraction, sphere formation and ALDH1 activity. More importantly, DATS inhibited cancer incidence and progression in 4-NQO oral carcinogenesis study. Further, DATS inhibited HNC tumor growth and HNC CSC fraction in vivo. Thus, DATS could be a potential chemopreventive and chemotherapeutic agent against head and neck cancer. Citation Format: Sivapar V. Mathan, Su-Hyeong Kim, Shivendra V. Singh, Rana P. Singh. Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2583.

RAD51AP1 Loss Attenuates Colorectal Cancer Stem Cell Renewal and Sensitizes to Chemotherapy.

DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)-induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1-deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer (ApcMin /+ ). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment. IMPLICATIONS: This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal.

Transcriptomic mapping on Notch signaling in A luminal phenotype breast cancer.

e12539 Background: The Notch signaling pathway plays an important role in cell-differentiation, survival, proliferation, stem-cell renewal, and in determining cell fate during development and morphogenesis. The dysregulation of the Notch pathways contributes to carcinogenesis, cancer stem cell renewal, angiogenesis, and chemo-resistance. Elevated levels of Notch receptors and ligands have been associated with cancer-progression and poor survival. A less explored function of Notch pathways in cancer is their role in leukocyte homing and activation. Understanding their role and relationship with immune infiltrates is an area of interest in cancer research. Methods: The expression of four different Notch genes (Notch1, Notch2, Notch3 and Notch4) was explored in relation with A luminal breast cancer patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to explore the association of the identified notch and immune infiltration, and the TCGA and METABRIC studies to analyze the correlation between notch1 - 4 expression and genomic signatures of immune activation. Results: We identified 2 individual genes called Notch1 and Notch2, which predict favorable prognosis in luminal A breast cancer. Their expression positively correlated with the presence of immune infiltrates within the tumor (dendritic cells, CD4+ T cells, neutrophils, CD8+ T cells and B cells), with markers of T cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast Notch3 and Notch4 which predicted for detrimental outcome were not associated with any of these parameters. Conclusions: Our analysis identifies a Notch signature composed of 2 genes with potential to recognize immune infiltrated and activated A luminal phenotype breast cancers with favorable prognosis.

A Systems Approach Identifies Key Regulators of HPV-Positive Cervical Cancer

Cervical cancer has remained the most prevalent and lethal malignancy among women worldwide and accounted for over 250,000 deaths in 2019. Nearly ninety-five per cent of cervical cancer cases are associated with persistent infection with high-risk Human Papillomavirus (HPV), and seventy per cent of these are associated with viral integration in the host genome. HPV-infection imparts specific changes in the regulatory network of infected cancer cells that are of diagnostic, prognostic and importance. Here, we conducted a systems-level analysis of the regulatory network changes, and the associated regulatory proteins thereof, in HPV-positive cervical cancer. We applied functional pathway analysis to show that HPV-positive cancers are characterised by perturbations of numerous cellular processes, predominantly in those linked to the cell cycle, mitosis, cytokine and immune cell signalling. Using computational predictions, we revealed that HPV-positive cervical cancers are regulated by transcription factors including, SOX2, E2F, NANOG, OCT4, and MYC, which control various processes such as the renewal of cancer stem cells, and the proliferation and differentiation of tumour cells. Through the analysis of upstream regulatory kinases, we identified the mitogen-activated protein kinases; among others, MAPK1, MAPK3 and MAPK8, and the cyclin-dependent kinases; among others, CDK1, CDK2 and CDK4, as the key kinases that control the biological processes in HPV-positive cervical cancers. Taken together, we uncover a landscape of the key regulatory pathways and proteins in HPV-positive cervical cancers, all of which may provide attractive drug targets for future therapeutics.