Sustained Cell Proliferation Research Articles

Obesity-Associated Colorectal Cancer.

Colorectal cancer (CRC) affects approximately 2 million people worldwide. Obesity is the major risk factor for CRC. In addition, obesity contributes to a chronic inflammatory stage that enhances tumor progression through the secretion of proinflammatory cytokines. In addition to an increased inflammatory response, obesity-associated cancer presents accrued molecular factors related to cancer characteristics, such as genome instability, sustained cell proliferation, telomere dysfunctions, angiogenesis, and microbial alteration, among others. Despite the evidence accumulated over the last few years, the treatments for obesity-associated CRC do not differ from the CRC treatments in normal-weight individuals. In this review, we summarize the current knowledge on obesity-associated cancer, including its epidemiology, risk factors, molecular factors, and current treatments. Finally, we enumerate possible new therapeutic targets that may improve the conditions of obese CRC patients. Obesity is key for the development of CRC, and treatments resulting in the reversal of obesity should be considered as a strategy for improving antineoplastic CRC therapies.

Comprehensive evaluation of ethanol-preserved amniotic extracts: Exploring antioxidant properties, proliferation enhancement, protective efficacy and regeneration potential in wound healing

Amniotic membrane extract (AME) has significant regenerative potential, which has been preserved using various methods over the years. This study explores ethanol (ETOH) as a preservative for AME in regenerative medicine. The study evaluated ETOH-preserved AME at 70 % (ETOH 70 %) and 50 % (ETOH 50 %) concentrations stored at room temperature (RT) and −80 °C. These were compared to phosphate buffer saline (PBS) and Dulbecco's Modified Eagle Medium/Glycerol (DMEM/GLY). In vitro assays included MTT for proliferation, apoptosis assays for protective effects, and scratch assays for migration using adipose stromal cells (ASCs) and fibroblasts. In vivo assessments involved subcutaneous injections in rats, with wound size measured at 4-, 8-, and 11-days post-injury. Histopathological parameters were analyzed after 11 days to evaluate tissue healing and regeneration.The results showed that ETOH preservation, particularly at 50 % and 70 %, exhibited significant antioxidant potential for AME, outperforming traditional methods. ETOH-preserved AME inhibited reactive oxygen species (ROS) and enhanced proliferation rates in ASCs compared to DMEM/GLY (p < 0.01). The ETOH 50 % variant at RT significantly reduced apoptosis and sustained cell proliferation. In vivo, ETOH-preserved AME at 50 % and 70 % accelerated wound closure significantly compared to the control group (p < 0.05). Histopathological analysis revealed improved healing, collagen deposition, and tissue regeneration in treated groups. ETOH preservation is a viable and cost-effective method for maintaining the regenerative potential of AME. This approach offers notable antioxidant, proliferative, and cytoprotective benefits, suggesting its promise for diverse biomedical applications.

Manipulating mannose metabolism as a potential anticancer strategy.

Cancer cells acquire metabolic advantages over their normal counterparts regarding the use of nutrients for sustained cell proliferation and cell survival in the tumor microenvironment. Notable among the metabolic traits in cancer cells is the Warburg effect, which is a reprogrammed form of glycolysis that favors the rapid generation of ATP from glucose and the production of biological macromolecules by diverting glucose into various metabolic intermediates. Meanwhile, mannose, which is the C-2 epimer of glucose, has the ability to dampen the Warburg effect, resulting in slow-cycling cancer cells that are highly susceptible to chemotherapy. This anticancer effect of mannose appears when its catabolism is compromised in cancer cells. Moreover, de novo synthesis of mannose within cancer cells has also been identified as a potential target for enhancing chemosensitivity through targeting glycosylation pathways. The underlying mechanisms by which alterations in mannose metabolism induce cancer cell vulnerability are just beginning to emerge. This review summarizes the current state of our knowledge of mannose metabolism and provides insights into its manipulation as a potential anticancer strategy.

A Molecular Hypothesis on Malignant Transformation of Oral Lichen Planus: A Systematic Review and Meta-Analysis of Cancer Hallmarks Expression in This Oral Potentially Malignant Disorder.

We aimed to qualitatively and quantitatively analyze, through a systematic review and meta-analysis, the current evidence on the differential expression of the hallmarks of cancer in oral lichen planus (OLP) samples, in order to know the earliest molecular mechanisms that could be involved in the malignant transformation of this oral potentially malignant disorder. We searched MEDLINE/PubMed, Embase, Web of Science, and Scopus for studies published before November 2023. We evaluated the methodological quality of studies and carried out meta-analyses to fulfill our objectives. Inclusion criteria were met by 110 primary-level studies, with 7065 OLP samples, in which the expression of 104 biomarkers were analyzed through immunohistochemistry. Most OLP samples showed sustained cell proliferation signaling (65.48%, 95%CI = 51.87-78.02), anti-apoptotic pathways (55.93%, 95%CI = 35.99-75.0), genome instability (48.44%, 95%CI = 13.54-84.19), and tumor-promoting inflammation events (83.10%, 95%CI = 73.93-90.74). Concurrently, OLP samples also harbored tumor growth suppressor mechanisms (64.00%, 95%CI = 53.27-74.12). In conclusion, current evidence indicates that molecular mechanisms promoting hyperproliferative signaling, an antiapoptotic state with genomic instability, and an escape of epithelial cells from immune destruction, are developed in LP-affected oral mucosa. It is plausible that these events are due to the actions exerted by the chronic inflammatory infiltrate. Malignant transformation appears to be prevented by tumor suppressor genes, which showed consistent upregulation in OLP samples.

KDM2B is required for ribosome biogenesis and its depletion unequally affects mRNA translation.

KDM2B is a JmjC domain lysine demethylase, which promotes cell immortalization, stem cell self-renewal and tumorigenesis. Here we employed a multi-omics strategy to address its role in ribosome biogenesis and mRNA translation. These processes are required to sustain cell proliferation, an important cancer hallmark. Contrary to earlier observations, KDM2B promotes ribosome biogenesis by stimulating the transcription of genes encoding ribosome biogenesis factors and ribosomal proteins, particularly those involved in the biogenesis of the 40S ribosomal subunits. Knockdown of KDM2B impaired the assembly of the small and large subunit processomes, as evidenced by specific defects in pre-ribosomal RNA processing. The final outcome was a decrease in the rate of ribosome assembly and in the abundance of ribosomes, and inhibition of mRNA translation. The inhibition of translation was distributed unequally among mRNAs with different features, suggesting that mRNA-embedded properties influence how mRNAs interpret ribosome abundance. This study identified a novel mechanism contributing to the regulation of translation and provided evidence for a rich biology elicited by a pathway that depends on KDM2B, and perhaps other regulators of translation.

Circular RNAs in Cell Cycle Regulation of Cancers.

Cancer has been one of the most problematic health issues globally. Typically, all cancers share a common characteristic or cancer hallmark, such as sustaining cell proliferation, evading growth suppressors, and enabling replicative immortality. Indeed, cell cycle regulation in cancer is often found to be dysregulated, leading to an increase in aggressiveness. These dysregulations are partly due to the aberrant cellular signaling pathway. In recent years, circular RNAs (circRNAs) have been widely studied and classified as one of the regulators in various cancers. Numerous studies have reported that circRNAs antagonize or promote cancer progression through the modulation of cell cycle regulators or their associated signaling pathways, directly or indirectly. Mostly, circRNAs are known to act as microRNA (miRNA) sponges. However, they also hold additional mechanisms for regulating cellular activity, including protein binding, RNA-binding protein (RBP) recruitment, and protein translation. This review will discuss the current knowledge of how circRNAs regulate cell cycle-related proteins through the abovementioned mechanisms in different cancers.

Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract 1704: Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma

Abstract Uveal melanoma (UM) originates from melanocytes residing in the uveal tract of an eye. Despite effective local control, 50% of patients develop metastatic disease responding poorly to chemotherapy, targeted therapy, and immunotherapy. The molecular basis of events leading to initiation of UM is related to mutations in G alpha proteins GNAQ or GNA11, rendering them constitutively active and activating downstream PKC, ERK and AKT pathways sustaining cell proliferation. In our previous work, we showed that NCOA3 regulates proliferation of UM cells in vitro and the growth of UM xenografts in vivo downstream of GNAQ or GNA11. The critical role of NCOA3 in UM is manifested by transcriptional regulation of proliferation-related genes that are massively suppressed by NCOA3 knockout or knockdown. Here, we interrogated transcription factors that NCOA3 interacts with in UM to execute oncogenic programs and identified Melanocyte inducing transcription factor (MITF) as its critical partner. Chromatin immunoprecipitation (ChIP) using an NCOA3 recognizing antibody followed by sequencing was performed to determine NCOA3 binding sites in the human genome. The known transcription binding motifs within NCOA3 peaks were discovered using HOMER algorithm. Co-immunoprecipitation was performed to examine direct NCOA3 and MITF interaction. ChIP sequencing for MITF was performed to determine co-localization of NCOA3 and MITF at chromatin. Proliferation of UM cells after inhibition of MITF by either siRNA mediated knockdown or MITF inhibitor ML329 was evaluated using MTT assay. Functional enrichment analysis of transcriptome was used to identify classes of genes regulated by MITF, with specific attention to the subset regulated by MITF partnered with NCOA3. NCOA3 chromatin peaks are highly enriched in MITF binding motifs, and 77% of the NCOA3 peaks co-localizes with the MITF peaks. NCOA3 co-immunoprecipitates with MITF. Among transcriptional programs regulated by MITF in UM are those related to melanocyte biology, differentiation, and melanin production, but also those related to melanoma growth, especially cell division, DNA replication, myc response, purine and pyrimidine metabolism, and mTOR signaling. Strong suppression of these transcriptional signatures by inhibition of MITF using siRNA-mediated knockdown or pharmacological inhibition correlates with dramatic loss of cell viability. Clinical data indicate that higher expression of MITF-positively regulated genes correlates with lower survival rates of UM patients. Importantly, the transcriptional programs regulated by MITF cooperating with NCOA3 were related to TGF beta signaling, cell adhesion, ECM interaction and actin cytoskeleton. Importance of MITF as an oncogene is manifested in uveal melanoma cell lines and clinical data. Oncogenic function of MITF is partially dependent on its interaction with a transcription co-activator NCOA3. Citation Format: Aleksandra Rusin, Maria E. Echartea, Sai P. Manikonda, Darlene G. Skapura, Christel M. Davis, Erik A. Ehli, Sandra L. Grimm, Cristian Coarfa, Salma Kaochar. Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma [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 1704.

Siah2 inhibitor and the metabolic antagonist Oxamate retard colon cancer progression and downregulate PD1 expression.

Solid tumors such as colon cancer are characterized by rapid and sustained cell proliferation, which ultimately results in hypoxia, induction of hypoxia-inducible factor-1α (HIF-1α), and activation of glycolysis to promote tumor survival and immune evasion. We hypothesized that a combinatorial approach of menadione (MEN) as an indirect HIF-1α inhibitor and sodium oxamate (OX) as a glycolysis inhibitor may be a promising treatment strategy for colon cancer. We investigated the potential efficacy of this combination for promoting an antitumor immune response and suppressing tumor growth in a rat model of colon cancer. Colon cancer was induced by once-weekly subcutaneous injection of 20 mg/kg dimethylhydrazine (DMH) for 16 weeks. Control rats received the vehicle and then no further treatment (negative control) or MEN plus OX for 4 weeks (drug control). Dimethylhydrazine-treated rats were then randomly allocated to four groups: DMH alone group and other groups treated with MEN, OX, and a combination of (MEN and OX) for 4 weeks. Serum samples were assayed for the tumor marker carbohydrate antigen (CA19.9), while expression levels of HIF-1α, caspase-3, PHD3, LDH, and PD1 were evaluated in colon tissue samples by immunoassay and qRT-PCR. Additionally, Ki-67 and Siah2 expression levels were examined by immunohistochemistry. The combination of MEN plus OX demonstrated a greater inhibitory effect on the expression levels of HIF-1α, Siah2, LDH, Ki-67, and PD1, and greater enhancement of caspase-3 and PHD3 expression in colon cancer tissues than either drug alone. Simultaneous targeting of hypoxia and glycolysis pathways by a combination of MEN and OX could be a promising therapy for inhibiting colon cancer cell growth and promoting antitumor immunity [1].

Diosgenin potentiates the anticancer effect of doxorubicin and volasertib via regulating polo-like kinase 1 and triggering apoptosis in hepatocellular carcinoma cells.

A common approach to cancer therapy is the combination of a natural product with chemotherapy to overcome sustained cell proliferation and chemotherapy resistance obstacles. Diosgenin (DG) is a phytosteroidal saponin that is naturally present in a vast number of plants and has been shown to exert anti-cancer activities against several tumor cells. Herein, we assessed the chemo-modulatory effects of DG on volasertib (Vola) as apolo-like kinase 1 (PLK1) inhibitor and doxorubicin (DOX) in hepatocellular carcinoma (HCC) cell lines. DOX and Vola were applied to two human HCC cell lines (HepG2 and Huh-7) alone or in combination with DG. The cell viability was determined, and gene expressions of PLK1, PCNA, P53, caspase-3, and PARP1 were evaluated by RT-qPCR. Moreover, apoptosis induction was determined by measuring active caspase-3 level using ELISA method. DG enhanced the anticancer effects of Vola and DOX. Moreover, DG enhanced Vola- and DOX-induced cell death by downregulating the expressions of PLK1 and PCNA, elevating the expressions of P53 and active caspase-3. DG showed promising chemo-modulatory effects to Vola and DOX against HCC that may be attributed partly to the downregulation of PLK1 and PCNA, upregulation of tumor suppressor protein P53, and apoptosis induction. Thus,DG combination with chemotherapy may be a promising treatment approach for HCC.

Artificial Modulation and Rewiring of Cell Cycle Progression Using Synthetic Circuits in Fission Yeast.

Cell cycle control is a central aspect of the biology of proliferating eukaryotic cells. However, progression through the cell cycle relies on a highly complex network, making it difficult to unravel the core design principles underlying the mechanisms that sustain cell proliferation and the ways in which they interact with other cellular pathways. In this context, the use of a synthetic approach to simplify the cell cycle network in unicellular genetic models such as fission yeast has opened the door to studying the biology of proliferating cells from unique perspectives. Here, we provide a series of methods based on a minimal cell cycle module in the fission yeast Schizosaccharomyces pombe that allows for an unprecedented artificial control of cell cycle events, enabling the rewiring and remodeling of cell cycle progression.

Switch-Independent 3A: An Epigenetic Regulator in Cancer with New Implications for Pulmonary Arterial Hypertension.

Epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNA, play a crucial role in the regulation of gene expression and are pivotal in biological processes like apoptosis, cell proliferation, and differentiation. SIN3a serves as a scaffold protein and facilitates interactions with transcriptional epigenetic partners and specific DNA-binding transcription factors to modulate gene expression by adding or removing epigenetic marks. However, the activation or repression of gene expression depends on the factors that interact with SIN3a, as it can recruit both transcriptional activators and repressors. The role of SIN3a has been extensively investigated in the context of cancer, including melanoma, lung, and breast cancer. Our group is interested in defining the roles of SIN3a and its partners in pulmonary vascular disease. Pulmonary arterial hypertension (PAH) is a multifactorial disease often described as a cancer-like disease and characterized by disrupted cellular metabolism, sustained vascular cell proliferation, and resistance to apoptosis. Molecularly, PAH shares many common signaling pathways with cancer cells, offering the opportunity to further consider therapeutic strategies used for cancer. As a result, many signaling pathways observed in cancer were studied in PAH and have encouraged new research studying SIN3a's role in PAH due to its impact on cancer growth. This comparison offers new therapeutic options. In this review, we delineate the SIN3a-associated epigenetic mechanisms in cancer and PAH cells and highlight their impact on cell survival and proliferation. Furthermore, we explore in detail the role of SIN3a in cancer to provide new insights into its emerging role in PAH pathogenesis.

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.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank &amp;lt; 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

Harnessing Human Placental Membrane-Derived Bioinks: Characterization and Applications in Bioprinting and Vasculogenesis.

Bioprinting applications in the clinical field generate great interest, but developing suitable biomaterial inks for medical settings is a challenge. Placental tissues offer a promising solution due to their abundance, stability, and status as medical waste. They contain basement membrane components, have a clinical history, and support angiogenesis. This study formulates bioinks from two placental tissues, amnion (AM) and chorion (CHO), and compares their unique extracellular matrix (ECM) and growth factor compositions. Rheological properties of the bioinks are evaluated for bioprinting and maturation of human endothelial cells. Both AM and Cho-derived bioinks sustained human endothelial cell viability, proliferation, and maturation, promoting optimal vasculogenesis. These bioinks derived from human sources have significant potential for tissue engineering applications, particularly in supporting vasculogenesis. This research contributes to the advancement of tissue engineering and regenerative medicine, bringing everyone closer to clinically viable bioprinting solutions using placental tissues as valuable biomaterials.

ATM inhibition blocks glucose metabolism and amplifies the sensitivity of resistant lung cancer cell lines to oncogene driver inhibitors

BackgroundATM is a multifunctional serine/threonine kinase that in addition to its well-established role in DNA repair mechanisms is involved in a number of signaling pathways including regulation of oxidative stress response and metabolic diversion of glucose through the pentose phosphate pathway. Oncogene-driven tumorigenesis often implies the metabolic switch from oxidative phosphorylation to glycolysis which provides metabolic intermediates to sustain cell proliferation. The aim of our study is to elucidate the role of ATM in the regulation of glucose metabolism in oncogene-driven cancer cells and to test whether ATM may be a suitable target for anticancer therapy.MethodsTwo oncogene-driven NSCLC cell lines, namely H1975 and H1993 cells, were treated with ATM inhibitor, KU55933, alone or in combination with oncogene driver inhibitors, WZ4002 or crizotinib. Key glycolytic enzymes, mitochondrial complex subunits (OXPHOS), cyclin D1, and apoptotic markers were analyzed by Western blotting. Drug-induced toxicity was assessed by MTS assay using stand-alone or combined treatment with KU55933 and driver inhibitors. Glucose consumption, pyruvate, citrate, and succinate levels were also analyzed in response to KU55933 treatment. Both cell lines were transfected with ATM-targeted siRNA or non-targeting siRNA and then exposed to treatment with driver inhibitors.ResultsATM inhibition deregulates and inhibits glucose metabolism by reducing HKII, p-PKM2Tyr105, p-PKM2Ser37, E1α subunit of pyruvate dehydrogenase complex, and all subunits of mitochondrial complexes except ATP synthase. Accordingly, glucose uptake and pyruvate concentrations were reduced in response to ATM inhibition, whereas citrate and succinate levels were increased in both cell lines indicating the supply of alternative metabolic substrates. Silencing of ATM resulted in similar changes in glycolytic cascade and OXPHOS levels. Furthermore, the driver inhibitors amplified the effects of ATM downregulation on glucose metabolism, and the combined treatment with ATM inhibitors enhanced the cytotoxic effect of driver inhibitors alone by increasing the apoptotic response.ConclusionsInhibition of ATM reduced both glycolytic enzymes and OXPHOS levels in oncogene-driven cancer cells and enhanced apoptosis induced by driver inhibitors thus highlighting the possibility to use ATM and the driver inhibitors in combined regimens of anticancer therapy in vivo.

CD98 heavy chain as a prognostic biomarker and target for cancer treatment.

The SLC3A2 gene encodes for a cell-surface transmembrane protein CD98hc (4F2). CD98hc serves as a chaperone for LAT1 (SLC7A5), LAT2 (SLC7A8), y+LAT1 (SLC7A7), y+LAT2 (SLC7A6), xCT (SLC7A11) and Asc1 (SLC7A10) providing their recruitment to the plasma membrane. Together with the light subunits, it constitutes heterodimeric transmembrane amino acid transporters. CD98hc interacts with other surface molecules, such as extracellular matrix metalloproteinase inducer CD147 (EMMPRIN) and adhesion receptors integrins, and regulates glucose uptake. In this way, CD98hc connects the signaling pathways sustaining cell proliferation and migration, biosynthesis and antioxidant defense, energy production, and stem cell properties. This multifaceted role makes CD98hc one of the critical regulators of tumor growth, therapy resistance, and metastases. Indeed, the high expression levels of CD98hc were confirmed in various tumor tissues, including head and neck squamous cell carcinoma, glioblastoma, colon adenocarcinoma, pancreatic ductal adenocarcinoma, and others. A high expression of CD98hc has been linked to clinical prognosis and response to chemo- and radiotherapy in several types of cancer. In this mini-review, we discuss the physiological functions of CD98hc, its role in regulating tumor stemness, metastases, and therapy resistance, and the clinical significance of CD98hc as a tumor marker and therapeutic target.

Long Non-Coding RNAs as "MYC Facilitators".

In this article, we discuss a class of MYC-interacting lncRNAs (long non-coding RNAs) that share the following criteria: They are direct transcriptional targets of MYC. Their expression is coordinated with the expression of MYC. They are required for sustained MYC-driven cell proliferation, and they are not essential for cell survival. We refer to these lncRNAs as "MYC facilitators" and discuss two representative members of this class of lncRNAs, SNHG17 (small nuclear RNA host gene) and LNROP (long non-coding regulator of POU2F2). We also present a general hypothesis on the role of lncRNAs in MYC-mediated transcriptional regulation.

RBM4 dictates ESCC cell fate switch from cellular senescence to glutamine-addiction survival through inhibiting LKB1-AMPK-axis

Cellular senescence provides a protective barrier against tumorigenesis in precancerous or normal tissues upon distinct stressors. However, the detailed mechanisms by which tumor cells evade premature senescence to malignant progression remain largely elusive. Here we reported that RBM4 adversely impacted cellular senescence to favor glutamine-dependent survival of esophageal squamous cell carcinoma (ESCC) cells by dictating the activity of LKB1, a critical governor of cancer metabolism. The level of RBM4 was specifically elevated in ESCC compared to normal tissues, and RBM4 overexpression promoted the malignant phenotype. RBM4 contributed to overcome H-RAS- or doxorubicin-induced senescence, while its depletion caused P27-dependent senescence and proliferation arrest by activating LKB1-AMPK-mTOR cascade. Mechanistically, RBM4 competitively bound LKB1 to disrupt the LKB1/STRAD/MO25 heterotrimeric complex, subsequently recruiting the E3 ligase TRIM26 to LKB1, promoting LKB1 ubiquitination and degradation in nucleus. Therefore, such molecular process leads to bypassing senescence and sustaining cell proliferation through the activation of glutamine metabolism. Clinically, the ESCC patients with high RBM4 and low LKB1 have significantly worse overall survival than those with low RBM4 and high LKB1. The RBM4 high/LKB1 low expression confers increased sensitivity of ESCC cells to glutaminase inhibitor CB-839, providing a novel insight into mechanisms underlying the glutamine-dependency to improve the efficacy of glutamine inhibitors in ESCC therapeutics.

Abstract 2102: Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model

Abstract Esophageal squamous cell carcinoma (ESCC) metastatic-initiating cells are heterogenous subpopulations that are hypothesized to promote distal metastasis. Pulmonary metastatic human-origin ESCC mice model was established in our laboratory, chronological flow cytometry collection of tumor cells isolated from metastatic mice lungs at different timepoints inoculated via intravenous injection was followed by 10X genomics multiplex single-cell transcriptomic sequencing (scRNA-seq). Bioinformatics analyses denoted a 7-signature panel previously introduced with preliminary data, follow-up validations of signature-enriched subpopulation in silico, in vitro, and in vivo were performed in this study. In silico bulk transcriptomic sequencing (RNA-seq) of single signature-enriched KYSE30 subpopulation followed by gene ontology (GO) enrichment analyses indicated significantly (FDR&amp;lt;0.05) enhanced biological processes involved in cell motility, cell migration and cell adhesion, compared to signature-low subpopulation. In vitro western blotting showed elevated mesenchymal markers expression, while diminished epithelial markers expression. In vitro real-time qPCR revealed single signature-enriched subpopulation has co-enrichment of other signature marker RNAs compared to control group. Further in vitro functional assays demonstrated signature-enriched subpopulation possesses strengthened spheroid formation ability (p&amp;lt;0.05) and soft agar colony formation ability (p&amp;lt;0.05) as compared to signature-low subpopulation. In addition to previous preliminary data on early survival ability in vivo, signature-enriched subpopulation was significantly more favorable in sustained cell survival and proliferation, and formation of larger size of metastatic lesions in mice lungs at 2 months post-intravenous injection as illustrated by flow cytometry (p&amp;lt;0.05) and immunohistochemistry (IHC) staining analyses (p&amp;lt;0.05) respectively as compared to signature-low subpopulation. To validate the protein expression of signature markers, 6-colour multiplex IHC staining was performed on KYSE30 cell line and metastatic mice lungs collected at different timepoints. Multiplex IHC staining exhibited that these signature markers were expressed in subpopulation of cells as well as expressed on inoculated tumor cells in metastatic mice lungs with timeline pattern. In summary, signature-enriched subpopulation manifested the metastatic-initiating properties in ESCC via various in silico, in vitro, and in vivo experimental analyses. Further multiplex staining in clinical tissue microarray would be performed and analyzed to examine feasibility of adopting these signature markers for facilitating clinical diagnosis and molecular targeting of metastatic ESCC. Citation Format: Ching Ngar Wong, Yu Zhang, Beibei Ru, Kwong Yiu Lam, Hongyu Zhou, Xinyuan Guan. Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model [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 2102.