Osteosarcoma (OS) is an aggressive primary bone malignancy with peak incidence in children and adolescents. Despite current multimodal treatments, there has been little change in overall survival outcomes in the last two decades. The canonical Wnt/β-catenin pathway is known to be a critical pathway in OS progression. To better understand the molecular basis of OS and potentially provide target/s for new therapies or diagnostics, we investigated the relationship between β-catenin, more specifically, the transcriptionally active form of β-catenin, Activated β-Catenin (ABC), and OS progression. We previously reported an association between ABC and aggressive OS whereby, cellular/nuclear ABC levels, but not cellular/nuclear β-catenin levels, increase with the degree of aggressiveness. However, a direct role for ABC in promoting OS progression has not been shown. In order to directly determine the role/impact of ABC in OS progression, we generated a pEGFP-ABC fusion construct which simulates ABC's phosphorylation pattern. Transfection of pEGFP-ABC, pEGFP-β-catenin, or an empty vector (pEGFP) into OS cell lines showed that wnt pathway transcriptional activity in GFP-ABC-expressing cells was significantly higher than that in both GFP-β-catenin and empty-vector-transfected cells. We also show that the in vitro invasive potential of the pEGFP-ABC-transfected cells was significantly higher compared to both pEGFP-β-catenin and pEGFP-transfected cells. Immunohistochemistry of clinical pediatric OS specimens supported these findings, showing a significant correlation between high ABC levels and invasive disease. To the best of our knowledge, this is the first report that suggest that ABC drive transcriptional activity to enhance invasiveness in OS and could serve as a biomarker of aggressive or metastatic OS.
BACKGROUND:Epstein-Barr Virus (EBV), a potent viral carcinogen, plays a crucial role in the development of various malignancies. Among its proteins, EBV nuclear antigen-1 (EBNA1) stands out for its ability to modulate gene expression. In this study, we explored the impact of EBNA1 on the expression patterns of four cellular genes-Derlin1, ZEB1, CNN3, and PSMD10-in HeLa cells. MATERIALS AND METHODS:Three distinct categories of HeLa cells were established: EBNA1-Transfected Cells: These cells were transfected with the EBNA1 gene.Control Plasmid-Transfected Cells: These cells received transfection with a control plasmid.Non-Transfected Cells (Control Group): These cells were not subjected to any transfection. After RNA extraction, we employed real-time PCR to evaluate the transcriptional levels of four specific genes-Derlin 1, ZEB1, CNN3, and PSMD10-in each of the three cell groups. The Mann-Whitney U-test was subsequently utilized to compare means, and statistical significance was determined based on p-values below 0.05. Data were meticulously recorded in an Excel 2016 spreadsheet. RESULTS:The results demonstrated that HeLa cells transfected with the EBNA1 plasmid exhibited significantly increased expression levels of Derlin1 (p = 0.028) and PSMD10 (p = 0.028) genes compared to cells transfected with the control plasmid. However, the expression changes observed in CNN3 and ZEB1 were not statistically significant (p = 0.99 and p = 0.2, respectively). CONCLUSIONS:Our findings suggest that increase expression levels of Derlin1 and PSMD10 genes in HeLa cells by the EBV-EBNA1 might induce cancer cell survival and accelerates the development of cervical cancer (CC). However, to establish a conclusive link between EBV-EBNA1 and CC progression, further investigations are warranted.
BACKGROUND:Gastric cancer (GC) is a multifactorial disease with a high death rate due to the unknown mechanisms involved in the developing, progressing, and late diagnosing GC. Several cancers have been linked to Long non-coding RNAs (lncRNAs), including GC, through differential expression. They play a crucial role in tumorigenesis pathways as modulatory factors, making them intriguing clinical and diagnostic biomarkers for many malignancies. This study's objective is to compare the lncRNAs CBR3-AS1 and PCA3 expression levels in tumoral tissues to marginal tissues and the clinicopathological features of patients. METHODS AND RESULTS:100 GC patients' tumoral and marginal tissue samples from Tabriz's Valiasr Hospital were gathered for this case-control research. To determine the expression level of PCA3 and CBR3-AS1 lncRNAs in GC, total RNA was extracted, and the qRT-PCR technique was employed. Compared to adjacent marginal tissues, the tumor tissue of patients with GC showed a significant increase in the expression levels of PCA3 and CBR3-AS1 (P < 0.0001). The expression ratio of lncRNA CBR3-AS1 and PCA3 did not significantly correlate with clinicopathological variables. The ROC curve's findings lead to the conclusion that the genes lncRNAs PCA3 and CBR3-AS1, with AUC values of 0.68 and 0.79, respectively, suggest that they could play carcinogenic roles in GC and may act as moderate diagnostic biomarkers for GC. CONCLUSIONS:In GC, CBR3-AS1 and PCA3 may be utilized as therapeutic targets and prognostic biomarkers, respectively.
Site-directed mutagenesis is a basic molecular tool required for protein, RNA and plasmid engineering. For mutagenesis methods, an ideal goal is to reach the efficiency of 100%. Towards this goal, we have recently taken the first step by adopting an innovative strategy using primer pairs with 3'-overhangs, thereby developing P3 site-directed mutagenesis, with an average efficiency of ~50%. As the second step towards the ideal goal, we report here P3a site-directed mutagenesis with an efficiency reaching ~100%. We systematically evaluated this new method by engineering >100 point mutations and small deletions (or insertions) on >20 mammalian expression vectors encoding various epigenetic regulators and the spike protein of SARS-CoV-2. As all known mutagenesis methods are limited to point mutations and small deletions/insertions (up to a dozen nucleotides), a technical problem is how to carry out cassette mutagenesis for replacement, deletion or insertion of large DNA fragments. The high efficiency of P3a mutagenesis and the 'handshaking' feature of primer pairs with 3'-overhangs inspired us to adapt this new method for seamless cassette mutagenesis, including highly efficient epitope tagging and untagging, deletion of small or large DNA fragments (up to 5 kb) and insertion of gene fragments (up to ~0.4 kb), LoxP sites and sequences encoding degrons, sgRNA and tigRNA. Thus, this new site-specific and cassette mutagenesis method is highly efficient, fast and versatile, likely resulting in its wide use for typical biomedical research, as well as for engineering and refining synthetic or mutant proteins from AI-assisted design.
Retinoblastoma ( Rb1) is a gene that codes for a tumour suppressor protein involved in various types of cancer.It was first described in retinoblastoma and is segregated as an autosomal dominant trait with high penetrance.In 1971, Knudson proposed his hypothesis of the two hits, where two mutational events are required to initiate tumour progression.We analysed three different point mutations present in patients' retinoblastoma.We produced three cell lines with retinoblastoma protein (RB) mutated in various regions: the missense pN328H, pD718N, and the nonsense early stop codon pR552*.We studied the effect of these point mutations on levels of mRNA and protein expression, proliferation, viability, localisation, and migration using an RBKO cell line.All three affected their localisation patterns and proliferation.However, the pR552* mutation also increases viability and migration.Moreover, when this mutation is simultaneously expressed with a wild-type RB, the phenotype and proliferation parameters are as with the mutant alone, suggesting that maybe only one mutated allele is needed to trigger the characteristic cancer phenotype.In other words, the pR552* mutant behaves more like a gain-of-function or oncogenic mutant.Indeed, a family carrying this mutation showed complete penetrance and high expressivity.
The MYC gene is a regulatory and proto-oncogenic gene that is overexpressed in the majority of prostate cancers (PCa). Numerous studies have indicated that aberrant expression of microRNAs is involved in the initiation and progression of prostate cancer. In this investigation, we assessed the impact of miR-377 on MYC through luciferase assay. Real-time PCR was employed to determine whether miR-377 could reduce the levels of MYC mRNA in transfected PCa cell lines (PC-3 and DU145) and change in the mRNA levels of BCL-2/Bax, PTEN, and CDK4 as a consequence of MYC downregulation. Moreover, we analyzed the effects of miR-377 on apoptosis, proliferation, cell cycle, and wound healing. Our findings demonstrate that miR-377 effectively targets MYC mRNA, as confirmed by luciferase assay and Real-time PCR. We observed a significant reduction in BCL-2 and CDK4 expression, along with an increase in Bax and PTEN, in prostate cancer cell lines upon MYC suppression. Additionally, elevated levels of miR-377 in PCa cell lines induced apoptosis, inhibited proliferation and migration, and arrested the cell cycle. Taken together, these results unveil the inhibitory role of miR-377 in MYC function within PCa, thereby suggesting its potential as a therapeutic target for the treatment of this malignancy.
Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer worldwide but is often diagnosed at an advanced incurable stage. Yet, despite the urgent need for blood-based biomarkers for early detection, few studies capture ongoing biology to identify risk-stratifying biomarkers. We address this gap using the TGF-β pathway because of its biological role in liver disease and cancer, established through rigorous animal models and human studies. Using machine learning methods with blood levels of 108 proteomic markers in the TGF-β family, we found a pattern that differentiates HCC from non-HCC in a cohort of 216 patients with cirrhosis, which we refer to as TGF-β based Protein Markers for Early Detection of HCC (TPEARLE) comprising 31 markers. Notably, 20 of the patients with cirrhosis alone presented an HCC-like pattern, suggesting that they may be a group with as yet undetected HCC or at high risk for developing HCC. In addition, we found two other biologically relevant markers, Myostatin and Pyruvate Kinase M2 (PKM2), which were significantly associated with HCC. We tested these for risk stratification of HCC in multivariable models adjusted for demographic and clinical variables, as well as batch and site. These markers reflect ongoing biology in the liver. They potentially indicate the presence of HCC early in its evolution and before it is manifest as a detectable lesion, thereby providing a set of markers that may be able to stratify risk for HCC.
Ewing sarcoma is a cancer of bone and soft tissue in children and young adults that is driven by the EWS-ETS fusion transcription factor, most commonly EWS-FLI1.We previously reported that Ewing sarcoma harbors two populations of cells, the CD133 high population displaying higher growth rate and the CD133 low population displaying chemotherapy resistance.We now find that the ubiquitin-specific protease 1 (USP1) is a transcriptional target of the EWS-FLI1 fusion oncoprotein, expressed at high and low levels in the CD133 high and the CD133 low populations, respectively, and determines chemo-sensitivity.We also find that USP1 inhibits cdc42, increases EWS-FLI1 transcriptional output, and simulates Ewing sarcoma growth.We show that chemo-sensitization by USP1 is independent of cdc42.A pharmacological inhibitor of USP1 was able to activate cdc42 and inhibit Ewing sarcoma growth.These results uncover critical roles for USP1 in Ewing sarcoma, which regulates growth and chemosensitivity via distinct mechanisms.
Background: In some breast cancers, decreased estrogen-sulfotransferase (SULT1E1) and its inactivation caused by oxidative-stress lead to elevated E2 levels as well as hypoxia-inducible tissue-damaging factors. Methods: Here, matrix-metalloproteases (MMP2/9) activity and SULT1E1-HIF1α protein/gene expression (Western-blot/RTPCR) were assessed in human breast-cancers versus their adjacent-tissues. Oxidant-stress neutralizer, chalcone (α,β unsaturated ketone) and SULT1E1-inducer dialyl-sulfide (source garlic; Allium sativum) were tested to prevent cancer causing factors in rat, in-vitro and in-vivo model. The antioxidant-enzymres SOD1, catalase, GPx and LDH, and matrix-degenerating MMP2/9 activities were assessed (gel-zymogram). Histoarchitecture (HE-staining) and tissue SULT1E1-localization (immuno-histochemistry) were screened. Extensive statistical-analysis were performed. Results: Human cancer-tissue expresses higher SULT1E1, paralleling HIF1α protein/mRNA owing to lower LDH activity. In addition, increase of MMP2/9 activities commenced tissue damage. However, chalcone and DAS significantly induced SULT1E1 gene/protein, and suppressed HIF1α expression, and MMP2/9 activities in rat tissues. Correlation of individual parameter statistics and group statistics of t-test suggest significant correlation of oxidative-stress (MDA) with SULT1E1 (p=0.006), HIF1α (p=0.006) protein-expression. The NPSH showed a negative correlation (p=0.001) with HIF1α, These two proteins and SULT1E1 mRNA expressions in human breast tumor were significantly higher (p<0.05) compared to the adjacent tissues. Pearson correlation data suggest, SULT1E1 is correlated with NPSH in different exposure groups. Conclusions: Breast cancers associate with SULT1E1, HIF1α and MMPs deregulations. Higher SULT1E1-protein in advanced cancer, remain inactive in oxidant oxidative environment and may be re-activated in chalcone induced reducing-state. Moreover, DAS induced SULT1E1 mRNA expression augments its protein increment. Synergistic drug-effects commenced HIF1α and MMPs suppression. Further studies are necessary.
We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro. However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein (VCP) as an essential gene in KRAS-mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS-mutant PDAC.
The t (8; 21) (q22; q22) with the resulting RUNX1- RUNX1T1 rearrangement is one of the most common cytogenetic abnormalities in acute myeloid leukemia (AML). It is associated with favorable prognosis. The t (5; 17) (q35; q21) is an uncommon translocation, fuses the gene for the nucleophosmin (NPM) to the retinoic acid receptor α(RARA) and was described essentially in acute promyelocytic leukemia (APL) variant. We present the case of a 19-year-old male patient who developed an AML with t (8; 21) (q22; q22) associated to t (5; 17) (q35; 21). Morphology and immunophenotype of the leukemic cells were compatible with AML. The patient received chemotherapy based on cytarabine and anthracycline without all-trans retinoic acid (ATRA) followed by allogenic stem cell transplantation in first remission. To the best of our knowledge, this is the first report of an association between a rare translocation t (5; 17) and t (8; 21) in AML. In this report, we will discuss the prognosis of this association as well as the treatment.
Chemoresistance in ovarian carcinoma is a puzzling issue that urges understanding of strategies used by cancer cells to survive DNA damage and to escape cell death. Expanding efforts to understand mechanisms driving chemoresistance and to develop alternative therapies targeting chemoresistant tumors are critical. Amplification of BRD4 is frequently associated with chemoresistant ovarian carcinoma, but little is known about the biological effects of the overexpression of BRD4 isoforms in this malignancy. Here, we described the consequences of BRD4-L and BRD4-S overexpression in ovarian carcinoma shedding a light on a complex regulation of BRD4 isoforms. We demonstrated that the BRD4-L transcript expression is required to generate both isoforms, BRD4-L and BRD4-S. We showed that the BRD4-S mRNA expression positively correlated with BRD4-S protein levels, while BRD4-L isoform showed negative correlation between mRNA and protein levels. Moreover, we demonstrated that an overexpression of BRD4 isoforms is associated with chemoresistance in ovarian cancer.
The CEA family comprises 18 genes and 11 pseudogenes located at chromosome 19q13.2 and is divided into two main groups: cell surface anchored CEA-related cell adhesion molecules (CEACAMs) and the secreted pregnancy-specific glycoproteins (PSGs). CEACAMs are highly glycosylated cell surface anchored, intracellular, and intercellular signaling molecules with diverse functions, from cell differentiation and transformation to modulating immune responses associated with infection, inflammation, and cancer. In this review, we explore current knowledge surrounding CEACAM1, CEACAM5, and CEACAM6, highlight their pathological significance in the areas of cancer biology, immunology, and inflammatory disease, and describe the utility of murine models in exploring questions related to these proteins.
In recent years, immunotherapy has finally found its place in the anti-cancer therapeutic arsenal, even becoming standard of care as first line treatment for metastatic forms. The clinical benefit provided by checkpoint blockers such as anti-PD-1/PD-L1 in many cancers revolutionized the field. However, too many patients remain refractory to these treatments due to weak baseline anti-cancer immunity. There is therefore a need to boost the frequency and function of patients' cytotoxic CD8+ cellular effectors by targeting immunogenic and tumor-restricted antigens, such as neoantigens using an efficient vaccination platform. Dendritic cells (DC) are the most powerful immune cell subset for triggering cellular immune response. However, autologous DC-based vaccines display several limitations, such as the lack of reproducibility and the limited number of cells that can be manufactured. Here we discuss the advantages of a new therapeutic vaccine based on an allogeneic Plasmacytoid DC cell line, which is easy to produce and represents a powerful platform for priming and expanding anti-neoantigen cytotoxic CD8+ T-cells.
Tumor-associated inflammation and chromosomal aberrations can play crucial roles in cancer development and progression. In neuroblastoma (NB), the enzyme cyclooxygenase-2 (COX-2) is associated with copy number alterations on the long arm of chromosome 11 (Ch 11q), defining an aggressive disease subset. This retrospective study included formalin-fixed paraffin-embedded tumor samples collected from nine patients during diagnosis at the pediatric Pequeno Principe Hospital, Curitiba, PR, Brazil, and post-chemotherapy (CT). COX-2 expression was evaluated using immunohistochemistry and correlated with the genome profile of paired pre- and post-CT samples, determined by array comparative genomic hybridization. A systems biology approach elucidated the PTGS2 network interaction. The results showed positive correlations between pre-CT Ch 7q gain and COX-2 expression (ρ = 0.825; p-value = 0.006) and negative correlations between Ch 7q gain and Ch 11q deletion (ρ = −0.919; p-value = 0.0005). Three samples showed Ch 11q deletion and Ch 7q gain. Network analysis identified a direct connection between CAV-1 (Ch 7q) and COX-2 in NB tumors and highlighted the connection between amplified genes in Ch 7q and deleted ones in 11q. The identification of hub-bottleneck-switch genes provides new biological insights into this connection between NB, tumorigenesis, and inflammation.
Hepatocellular carcinoma (HCC) is the primary form of liver cancer and a major cause of cancer death worldwide.Early detection is key to effective treatment.Yet, early diagnosis is challenging, especially in patients with cirrhosis, who are at high risk of developing HCC.Dysfunction or loss of function of the transforming growth factor β (TGF-β) pathway is associated with HCC.Here, using quantitative immunohistochemistry analysis of samples from a multi-institutional repository, we evaluated if differences in TGF-β receptor abundance were present in tissue from patients with only cirrhosis compared with those with HCC in the context of cirrhosis.We determined that TGFBR2, not TGFBR1, was significantly reduced in HCC tissue compared with cirrhotic tissue.We developed an artificial intelligence (AI)-based process that correctly identified cirrhotic and HCC tissue and confirmed the significant reduction in TGFBR2 in HCC tissue compared with cirrhotic tissue.Thus, we propose that a reduction in TGFBR2 abundance represents a useful biomarker for detecting HCC in the context of cirrhosis and that incorporating this biomarker into an AI-based automated imaging pipeline could reduce variability in diagnosing HCC from biopsy tissue.
Genes are transcribed to produce pre-mRNAs, which are then spliced to create the mature mRNAs translated into protein. In recent years, improved deep sequencing technologies have shown greater than 90% of human pre-mRNAs undergo alternative splicing, thereby amplifying the potential protein products from each gene [1]. Alternatively spliced forms of pre-mRNA may code for proteins with related, distinct, or even opposing functions [1]. Many growth factor and hormone receptors and signaling molecules implicated in cancer have natural splice variants, some of which have been shown to act as dominant negatives. We hypothesized that by altering splicing to decrease growth-promoting and/or increase expression of dominant negative varieties we could eliminate abnormal dependence on growth factors, decrease metastatic potential, and promote cancer cell death. By binding to specific intronic or exonic regions or intron-exon junctions, splice modulating oligomers, which are cDNA sequences, can alter the outcome of splicing [e.g., 2, 3]. To our knowledge, no one had previously tapped the potential of splice modulating oligomers to increase the relative activity of natural dominant negatives in order to combat disease. Where splice modulating oligomers had begun to be explored as therapeutics was for diseases that result from splicing errors and the production of a non-functional protein [4, 5]. Dominant negative receptors may inhibit signaling from the growth-promoting form of the receptor in a variety of ways. In the simplest situation, a dominant negative receptor binds ligand and therefore reduces availability to the growth-promoting receptor. In other instances, the dominant negative receptor may generate an alternate intracellular signal [e.g., 6–9]. Such amplification of the effect of dominant negative receptors through a signaling cascade makes an increase in their relative expression all the more effective. Importantly and additionally, the signals generated can promote differentiation and/or apoptotic cell death [6–8], thereby Editorial
Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second most deadly cancer in the United States by 2030 [1]. Although stage 1A PDAC 5-year survival is now greater than 80%, the majority of PDAC is diagnosed at more advanced stages, with 5-year survival being less than 10% for stage III/IV disease [2]. The majority of PDAC is sporadic, however up to 10% of PDAC is considered to be familial [3], including individuals with a pathogenic or likely pathogenic variant (PV) in a known PDAC susceptibility gene and/or familial pancreatic cancer, defined as a family with at least two relatives with PDAC who are directly related to one another without known genetic susceptibility. As surgical resection of early-stage disease offers the highest chance of long-term survival, effective pancreatic cancer surveillance in high-risk individuals (HRIs) is imperative to allow for early detection. While some reports have not shown strong evidence that pancreatic cancer surveillance is effective [4, 5], older [6] as well as more recent data [7] from the Cancer of the Pancreas Screening (CAPS) studies showed that only 5% of surveillance detected PDACs in HRIs were stage IV, while 86% of PDACs diagnosed outside of surveillance were stage IV. Furthermore, 58% of surveillance detected PDACs were stage I, and 5-year survival amongst surveillance detected PDACs was 73% (median overall survival of 9.8 years vs. 1.5 years among HRIs with PDACs detected within and outside of surveillance, respectively) [7]. Notably though, only 26 (1.5%) PDAC cases were diagnosed amongst 1,731 patients enrolled in the CAPS studies [7]. Nonetheless, this data illustrates that PDAC surveillance of HRIs may lead to diagnosis of PDAC at earlier stages with improved long-term survival. PDAC surveillance guidelines for HRIs carrying a PV in a PDAC risk gene such as BRCA1, BRCA2, PALB2, ATM, and genes associated with Lynch syndrome have classically required a family history of PDAC in a first or second degree relative to qualify for surveillance [8]. This contrasts with carriers of higher risk PVs in genes such as CDKN2A and STK11 (lifetime PDAC risk > 15% [8]), where a family history of PDAC is not required for surveillance eligibility. For carriers of a BRCA1, BRCA2, PALB2, ATM, or Lynch syndrome PV, restricting PDAC surveillance to those with a family history of PDAC has Editorial
Ewing sarcoma is a cancer of bone and soft tissue in children driven by EWS::ETS fusion, most commonly EWS::FLI1. Because current cytotoxic chemotherapies are not improving the survival of those with metastatic or recurrent Ewing sarcoma cases, there is a need for novel and more effective targeted therapies. While EWS::FLI1 is the major driver of Ewing sarcoma, EWS::FLI1 has been difficult to target. A promising alternative approach is to identify and target the molecular vulnerabilities created by EWS::FLI1. Here we report that EWS::FLI1 induces the expression of Slit2, the ligand of Roundabout (Robo) receptors implicated in axon guidance and multiple other developmental processes. EWS::FLI1 binds to the Slit2 gene promoter and stimulates the expression of Slit2. Slit2 inactivates cdc42 and stabilizes the BAF chromatin remodeling complexes, enhancing EWS::FLI1 transcriptional output. Silencing of Slit2 strongly inhibited anchorage-dependent and anchorage-independent growth of Ewing sarcoma cells. Silencing of Slit2 receptors, Robo1 and Robo2, inhibited Ewing sarcoma growth as well. These results uncover a new role for Slit2 signaling in stimulating Ewing sarcoma growth and suggest that this pathway can be targeted therapeutically.
