Event In Tumor Development Research Articles

Loss of miR-101-3p in melanoma stabilizes genomic integrity, leading to cell death prevention

Malignant melanoma remains the most lethal form of skin cancer, exhibiting poor prognosis after forming distant metastasis. Owing to their potential tumor-suppressive properties by regulating oncogenes and tumor suppressor genes, microRNAs are important player in melanoma development and progression. We defined the loss of miR-101-3p expression in melanoma cells compared with melanocytes and melanoblast-related cells as an early event in tumor development and aimed to understand the tumor suppressive role of miR-101-3p and its regulation of important cellular processes. Reexpression of miR-101-3p resulted in inhibition of proliferation, increase in DNA damage, and induction of apoptosis. We further determined the nuclear structure protein Lamin B1, which influences nuclear processes and heterochromatin structure, ATRX, CASP3, and PARP as an important direct target of miR-101-3p. RNA sequencing and differential gene expression analysis after miR-101-3p reexpression supported our findings and the importance of loss of mir-101-3p for melanoma progression. The validated functional effects are related to genomic instability, as recent studies suggest miRNAs plays a key role in mediating this cellular process. Therefore, we concluded that miR-101-3p reexpression increases the genomic instability, leading to irreversible DNA damage, which leads to apoptosis induction. Our findings suggest that the loss of miR-101-3p in melanoma serves as an early event in melanoma progression by influencing the genomic integrity to maintain the increased bioenergetic demand.

Downregulation of ADAMTS3 Suppresses Stemness and Tumorigenicity in Glioma Stem Cell.

Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models. ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets. ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM. We identified ADAMTS3 as a potential therapeutic target for GBM.

Long-term treatment of cancer-prone germline PTEN mutant micewith low-dose rapamycin extends lifespan and delays tumour development.

PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underlie PTEN hamartoma tumour syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralise the impact of PTEN loss, rendering this a more druggable context compared with those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6 weeks onwards to mice with heterozygous germline Pten loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay in, but not full blockade of, the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

WTAP mediates FOXP3 mRNA stability to promote SMARCE1 expression and augment glycolysis in colon adenocarcinoma.

N6-methyladenosine (m6A) is the most abundant mRNA internal modification and has reportedly been linked to aerobic glycolysis, a hallmark event in tumor development. This work focuses on the role of the m6A methyltransferase WT1-associated protein (WTAP) in metabolic reprogramming and development of colon adenocarcinoma (COAD) and the molecules involved. The WTAP expression in COAD tissues and cells was detected. WTAP was knocked down in two COAD cell lines to figure out its role in the glycolytic activity and malignant phenotype of cancer cells. Cancer cells were further injected into nude mice subcutaneously or via tail vein to evaluate tumor growth and metastasis. The downstream molecules involved were explored using bioinformatics tools, and the molecular interactions were confirmed by immunoprecipitation, luciferase assays, and rescue experiments. WTAP was abundantly expressed in COAD samples. Knockdown of WTAP suppressed glucose consumption, lactate production, and glycolysis, which consequently suppressed cancer cell growth and dissemination in vitro and in vivo. WTAP promoted m6A methylation and stabilized forkhead box P3 (FOXP3) mRNA with the participation of the m6A "reader" YTHDF1. FOXP3 could further bind to SMARCE1 promoter for transcriptional activation. Rescue experiments showed that upregulation of FOXP3 or SMARCE1 restored the glycolytic activity in COAD cells and augmented the growth and mobility of cells both in vitro and in vivo. This study demonstrates that WTAP grants glycolytic activity to COAD and promotes tumor malignant development via the m6A modification of FOXP3 mRNA and the upregulation of SMARCE1.

HER2 Mediates PSMA/mGluR1-Driven Resistance to the DS-7423 Dual PI3K/mTOR Inhibitor in PTEN Wild-type Prostate Cancer Models.

Prostate cancer remains a major cause of male mortality. Genetic alteration of the PI3K/AKT/mTOR pathway is one of the key events in tumor development and progression in prostate cancer, with inactivation of the PTEN tumor suppressor being very common in this cancer type. Extensive evaluation has been performed on the therapeutic potential of PI3K/AKT/mTOR inhibitors and the resistance mechanisms arising in patients with PTEN-mutant background. However, in patients with a PTEN wild-type phenotype, PI3K/AKT/mTOR inhibitors have not demonstrated efficacy, and this remains an area of clinical unmet need. In this study, we have investigated the response of PTEN wild-type prostate cancer cell lines to the dual PI3K/mTOR inhibitor DS-7423 alone or in combination with HER2 inhibitors or mGluR1 inhibitors. Upon treatment with the dual PI3K/mTOR inhibitor DS-7423, PTEN wild-type prostate cancer CWR22/22RV1 cells upregulate expression of the proteins PSMA, mGluR1, and the tyrosine kinase receptor HER2, while PTEN-mutant LNCaP cells upregulate androgen receptor and HER3. PSMA, mGluR1, and HER2 exert control over one another in a positive feedback loop that allows cells to overcome treatment with DS-7423. Concomitant targeting of PI3K/mTOR with either HER2 or mGluR1 inhibitors results in decreased cell survival and tumor growth in xenograft studies. Our results suggest a novel therapeutic possibility for patients with PTEN wild-type PI3K/AKT-mutant prostate cancer based in the combination of PI3K/mTOR blockade with HER2 or mGluR1 inhibitors.

Histone Modifying Enzymes in Gynaecological Cancers.

Simple SummaryEpigenetics is a process that allows genetic control, without the involvement of sequence changes to DNA or genes. In cancer, epigenetics is a key event in tumour development that can alter the expression of cancer driver genes and result in genomic instability. Due to the critical role of epigenetics in malignant transformation, therapies that target these processes have been developed to treat cancer. Here, we provide a summary of the epigenetic changes that have been described in a variety of gynaecological cancers. We then highlight how these changes are being targeted in preclinical models and clinical trials for gynaecological cancers.Genetic and epigenetic factors contribute to the development of cancer. Epigenetic dysregulation is common in gynaecological cancers and includes altered methylation at CpG islands in gene promoter regions, global demethylation that leads to genome instability and histone modifications. Histones are a major determinant of chromosomal conformation and stability, and unlike DNA methylation, which is generally associated with gene silencing, are amenable to post-translational modifications that induce facultative chromatin regions, or condensed transcriptionally silent regions that decondense resulting in global alteration of gene expression. In comparison, other components, crucial to the manipulation of chromatin dynamics, such as histone modifying enzymes, are not as well-studied. Inhibitors targeting DNA modifying enzymes, particularly histone modifying enzymes represent a potential cancer treatment. Due to the ability of epigenetic therapies to target multiple pathways simultaneously, tumours with complex mutational landscapes affected by multiple driver mutations may be most amenable to this type of inhibitor. Interrogation of the actionable landscape of different gynaecological cancer types has revealed that some patients have biomarkers which indicate potential sensitivity to epigenetic inhibitors. In this review we describe the role of epigenetics in gynaecological cancers and highlight how it may exploited for treatment.

Divergent immune landscapes of primary and syngeneic Kras-driven mouse tumor models

Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. Both genetically engineered mouse models (GEMMs) and syngeneic cell transplant approaches use immunocompetent mice to define immune-dependent events in tumor development and progression. Due to their rapid and reproducible nature, there is expanded interest in developing new syngeneic tools from established primary tumor models. However, few studies have examined the extent that syngeneic tumors reflect the immune profile of their originating primary models. Here, we describe comprehensive immunophenotyping of two well-established GEMMs and four new syngeneic models derived from these parental primary tumors. To our knowledge, this is the first systematic analysis comparing immune landscapes between primary and orthotopic syngeneic tumors. These models all use the same well-defined human-relevant driver mutations, arise at identical orthotopic locations, and are generated in mice of the same background strain. This allows for a direct and focused comparison of tumor immune landscapes in carefully controlled mouse models. We identify key differences between the immune infiltrate of GEMM models and their corresponding syngeneic tumors. Most notable is the divergence of T cell populations, with different proportions of CD8+ T cells and regulatory T cells across several models. We also observe immune variation across syngeneic tumors derived from the same primary model. These findings highlight the importance of immune variance across mouse modeling approaches, which has strong implications for the design of rigorous and reproducible translational studies.

Chromothripsis in Human Breast Cancer.

Chromothripsis is a form of genome instability by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosomes. Widely assumed to be an early event in tumor development, this phenomenon plays a prominent role in tumor onset. In this study, an analysis of chromothripsis in 252 human breast cancers from two patient cohorts (149 metastatic breast cancers, 63 untreated primary tumors, 29 local relapses, and 11 longitudinal pairs) using whole-genome and whole-exome sequencing reveals that chromothripsis affects a substantial proportion of human breast cancers, with a prevalence over 60% in a cohort of metastatic cases and 25% in a cohort comprising predominantly luminal breast cancers. In the vast majority of cases, multiple chromosomes per tumor were affected, with most chromothriptic events on chromosomes 11 and 17 including, among other significantly altered drivers, CCND1, ERBB2, CDK12, and BRCA1. Importantly, chromothripsis generated recurrent fusions that drove tumor development. Chromothripsis-related rearrangements were linked with univocal mutational signatures, with clusters of point mutations due to kataegis in close proximity to the genomic breakpoints and with the activation of specific signaling pathways. Analyzing the temporal order of events in tumors with and without chromothripsis as well as longitudinal analysis of chromothriptic patterns in tumor pairs offered important insights into the role of chromothriptic chromosomes in tumor evolution. SIGNIFICANCE: These findings identify chromothripsis as a major driving event in human breast cancer.

Identification of Pre-Diagnostic Metabolic Patterns for Glioma Using Subset Analysis of Matched Repeated Time Points.

Simple SummaryReprogramming of cellular metabolism is a major hallmark of cancer cells, and play an important role in tumor initiation and progression. The aim of our study is to discover circulating early metabolic markers of brain tumors, as discovery and development of reliable predictive molecular markers are needed for precision oncology applications. We use a study design tailored to minimize confounding factors and a novel machine learning and visualization approach (SMART) to identify a panel of 15 interlinked metabolites related to glioma development. The presented SMART strategy facilitates early molecular marker discovery and can be used for many types of molecular data.Here, we present a strategy for early molecular marker pattern detection—Subset analysis of Matched Repeated Time points (SMART)—used in a mass-spectrometry-based metabolomics study of repeated blood samples from future glioma patients and their matched controls. The outcome from SMART is a predictive time span when disease-related changes are detectable, defined by time to diagnosis and time between longitudinal sampling, and visualization of molecular marker patterns related to future disease. For glioma, we detect significant changes in metabolite levels as early as eight years before diagnosis, with longitudinal follow up within seven years. Elevated blood plasma levels of myo-inositol, cysteine, N-acetylglucosamine, creatinine, glycine, proline, erythronic-, 4-hydroxyphenylacetic-, uric-, and aceturic acid were particularly evident in glioma cases. We use data simulation to ensure non-random events and a separate data set for biomarker validation. The latent biomarker, consisting of 15 interlinked and significantly altered metabolites, shows a strong correlation to oxidative metabolism, glutathione biosynthesis and monosaccharide metabolism, linked to known early events in tumor development. This study highlights the benefits of progression pattern analysis and provide a tool for the discovery of early markers of disease.

Comprehensive molecular comparison of BRCA1 hypermethylated and BRCA1 mutated triple negative breast cancers

Homologous recombination deficiency (HRD) is a defining characteristic in BRCA-deficient breast tumors caused by genetic or epigenetic alterations in key pathway genes. We investigated the frequency of BRCA1 promoter hypermethylation in 237 triple-negative breast cancers (TNBCs) from a population-based study using reported whole genome and RNA sequencing data, complemented with analyses of genetic, epigenetic, transcriptomic and immune infiltration phenotypes. We demonstrate that BRCA1 promoter hypermethylation is twice as frequent as BRCA1 pathogenic variants in early-stage TNBC and that hypermethylated and mutated cases have similarly improved prognosis after adjuvant chemotherapy. BRCA1 hypermethylation confers an HRD, immune cell type, genome-wide DNA methylation, and transcriptional phenotype similar to TNBC tumors with BRCA1-inactivating variants, and it can be observed in matched peripheral blood of patients with tumor hypermethylation. Hypermethylation may be an early event in tumor development that progress along a common pathway with BRCA1-mutated disease, representing a promising DNA-based biomarker for early-stage TNBC.

The landscape of chromothripsis across adult cancer types

Chromothripsis is a recently identified mutational phenomenon, by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosome(s). Considered as an early event in tumour development, this form of genome instability plays a prominent role in tumour onset. Chromothripsis prevalence might have been underestimated when using low-resolution methods, and pan-cancer studies based on sequencing are rare. Here we analyse chromothripsis in 28 tumour types covering all major adult cancers (634 tumours, 316 whole-genome and 318 whole-exome sequences). We show that chromothripsis affects a substantial proportion of human cancers, with a prevalence of 49% across all cases. Chromothripsis generates entity-specific genomic alterations driving tumour development, including clinically relevant druggable fusions. Chromothripsis is linked with specific telomere patterns and univocal mutational signatures in distinct tumour entities. Longitudinal analysis of chromothriptic patterns in 24 matched tumour pairs reveals insights in the clonal evolution of tumours with chromothripsis.

TERT promoter hotspot mutations and their relationship with TERT levels and telomere erosion in patients with head and neck squamous cell carcinoma.

To evaluate the prevalence of two recurrent somatic mutations(-124 C>T and -146 C>T) within thepromoterof the gene encoding telomerase reverse transcriptase (TERT) as well as their relationship with TERT level, telomeres length, and outcome in patients with head and neck squamous cell carcinomas (HNSCCs). We evaluate the prevalence of TERT promoter mutations, TERT levels, and telomere length in paired cancer tissue and adjacent mucosa (AM) in a series of HNSCCs. Cancer tissue and AM specimens from 105 patients were analyzed. Telomere length and TERT mRNA levels were estimated using real-time polymerase chain reaction. TERT promoter mutations were assessed using Sanger sequencing. Out of 105 cases, 101 were considered suitable for the analysis. TERT promoter harbored mutations in 12 tumors (11.9%), with -124 C>T and -146 C>T accounting for 83.3% and 16.7% of the alterations, respectively. No mutations were detected in AM samples. The prevalence of TERT promoter mutations was significantly higher in oral cavity SCCs (10 out of 27 tumors; 37%), and telomere length in AM was shorter in patients with tumors carrying TERT promoter mutations than in patients with unmutated TERT promoter cancers (p = 0.023). TERT levels in tumor did not significantly differ according to the mutational status of TERT promoter. No significant association was found between TERT promoter status and overall survival. TERT promoter mutations are most likely a late event in tumor development, occurring in a context of critically short telomeres, mostly in patients with oral cavity SCC. TERT levels, but not TERT promoter mutational status impact clinical outcome.

GENE-32. SYNTHETIC LETHAL INTERACTIONS WITH IDH1R132H IN GLIOMA STEM-LIKE CELLS

Abstract Gliomas are the most frequently diagnosed human primary brain tumors. Mutations in Isocitrate Dehydrogenase (IDH) 1 occur in the vast majority of low grade gliomas and secondary high grade glioblastomas. A single amino acid missense mutation in IDH1 at arginine 132 (R132H) is an early event in tumor development. IDH1R132H leads to the production of the oncometabolite 2-R-2-hydroxyglutarate. However the exact roles played by IDH1R132H in the development and malignant transformation of the tumors remain unclear. Further studies are required to determine optimal therapeutic strategies to target the IDH1 mutated subsets of gliomas. New generation high-throughput genetic perturbation technologies make it possible to systematically identify the genes and pathways required for the survival and proliferation of mammalian cells. Herein, we present preliminary results from a CRISPR-dCas9 derived activation to drive the transcriptional activation of more than 23,000 coding genes in both wild type and mutant IDH1 patient-derived pediatric glioma cells. Based on an average of three viability screens per cell type, we analyzed the sgRNA library representation in the IDH1 mutated and non-mutated glioma cultures after the genome wide activation. We identified 1553 candidate genes that upon gain of function trigger the death of glioma cells harboring the IDH1 mutation. The analysis of these results further pinpoints the activation of the Cyclin E1 (CCNE1), the BCL2 Antagonist/Killer 1 (BAK1) and the Homeobox B13 (HOXB13) as the most significant synthetic lethal targets in IDH1R132H glioma cells. Interestingly, results from RNAseq showed a decreased expression of these genes in IDH1 mutated compared to non-mutated glioma cells. Thus, this viability screening aims to elucidate genes that interact with IDH1R123H and play a role in tumor cell fitness. The functional analysis of these candidate genes will allow us to uncover their contribution to the progression of IDH1 mutated gliomas.

Effect of progesterone and first evidence about allopregnanolone action on the progression of epithelial human ovarian cancer cell lines

Ovarian carcinoma is one of the most common cause of death by gynecologic cancer. Several epidemiological and in vitro studies have shown controversial data about progesterone effects in ovarian cancer. Progesterone can be converted in its active metabolite, allopregnanolone, its effects in ovarian cancer are still unknown. Previously, we demonstrated that allopregnanolone modifies ovarian morphophysiology, being able to alter critical process of tumor development such as proliferation, apoptosis and angiogenesis. Taking into account these antecedents, we investigated the effect of progesterone and allopregnanolone on proliferation, apoptosis, clonogenic capacity and migration on two epithelial human ovarian cancer cell lines, IGROV-1 and SKOV-3. To this end, IGROV-1 and SKOV-3 cells were exposed to a range of progesterone and allopregnanolone concentrations (10-11 to 10-5 M) for 72 h. Proliferation was analyzed by MTT and Ki67 expression. Apoptosis was measured by immunocytochemistry of cleaved caspase 3. Clonogenic capacity was evaluated by counting colonies. Migration was analyzed by wound assay. We found that allopregnanolone increased proliferation and Ki67 expression respect to control on IGROV-1 cells, while expression of cleaved caspase 3 did not change in any cell line studied. IGROV-1 clonogenic capacity was also increased by allopregnanolone treatment. Both steroids, progesterone and allopregnanolone, increased IGROV-1 migration in a concentration dependent manner. None of the steroids tested modified SKOV-3 biological behavior analized. This is the first evidence that allopregnanolone, a progesterone metabolite, affects critical events in tumor development of human epithelial ovarian cancer. These results could have an impact in the future in clinic diagnosis, prognosis and treatment of ovarian cancer patients. The regulation of progesterone and allopregnanolone steroideogenesis and their molecular mechanisms might be considered as potential therapeutic tool in ovarian cancer.

Fusion-mediated chromosomal instability promotes aneuploidy patterns that resemble human tumors.

Oncogenesis is considered to result from chromosomal instability, in addition to oncogene and tumor-suppressor alterations. Intermediate to aneuploidy and chromosomal instability, genome doubling is a frequent event in tumor development but the mechanisms driving tetraploidization and its impact remain unexplored. Cell fusion, one of the pathways to tetraploidy, is a physiological process involved in mesenchymal cell differentiation. Besides simple genome doubling, cell fusion results in the merging of two different genomes that can be destabilized upon proliferation. By testing whether cell fusion is involved in mesenchymal oncogenesis, we provide evidence that it induces genomic instability and mediates tumor initiation. After a latency period, the tumor emerges with the cells most suited for its development. Furthermore, hybrid tumor genomes were stabilized after this selection process and were very close to those of human pleomorphic mesenchymal tumors. Thus genome restructuring triggered by cell fusion may account for the chromosomal instability involved in oncogenesis.

Angiogenesis in Pancreatic Cancer: Pre-Clinical and Clinical Studies.

Angiogenesis is a crucial event in tumor development and progression, occurring by different mechanisms and it is driven by pro- and anti-angiogenic molecules. Pancreatic cancer vascularization is characterized by a high microvascular density, impaired microvessel integrity and poor perfused vessels with heterogeneous distribution. In this review article, after a brief introduction on pancreatic cancer classification and on angiogenesis mechanisms involved in its progression, the pre-clinical and clinical trials conducted in pancreatic cancer treatment using anti-angiogenic inhibitors will be described. Finally, we will discuss the anti-angiogenic therapy paradox between the advantage to abolish vessel supply to block tumor growth and the disadvantage due to reduction of drug delivery at the same time. The purpose is to identify new anti-angiogenic molecules that may enhance treatment regimen.

Comprehensive characterization of the alternative splicing landscape in head and neck squamous cell carcinoma reveals novel events associated with tumorigenesis and the immune microenvironment.

Alternative splicing (AS) has emerged as a key event in tumor development and microenvironment formation. However, comprehensive analysis of AS and its clinical significance in head and neck squamous cell carcinoma (HNSC) is urgently required. Methods: Genome-wide profiling of AS events using RNA-Seq data from The Cancer Genome Atlas (TCGA) program was performed in a cohort of 464 patients with HNSC. Cancer-associated AS events (CASEs) were identified between paired HNSC and adjacent normal tissues and evaluated in functional enrichment analysis. Splicing networks and prognostic models were constructed using bioinformatics tools. Unsupervised clustering of the CASEs identified was conducted and associations with clinical, molecular and immune features were analyzed. Results: We detected a total of 32,309 AS events and identified 473 CASEs in HNSC; among these, 91 were validated in an independent cohort (n = 15). Functional protein domains were frequently altered, especially by CASEs affecting cancer drivers, such as PCSK5. CASE parent genes were significantly enriched in pathways related to HNSC and the tumor immune microenvironment, such as the viral carcinogenesis (FDR < 0.001), Human Papillomavirus infection (FDR < 0.001), chemokine (FDR < 0.001) and T cell receptor (FDR < 0.001) signaling pathways. CASEs enriched in immune-related pathways were closely associated with immune cell infiltration and cytolytic activity. AS regulatory networks suggested a significant association between splicing factor (SF) expression and CASEs and might be regulated by SF methylation. Eighteen CASEs were identified as independent prognostic factors for overall and disease-free survival. Unsupervised clustering analysis revealed distinct correlations between AS-based clusters and prognosis, molecular characteristics and immune features. Immunogenic features and immune subgroups cooperatively depict the immune features of AS-based clusters. Conclusion: This comprehensive genome-wide analysis of the AS landscape in HNSC revealed novel AS events related to carcinogenesis and immune microenvironment, with implications for prognosis and therapeutic responses.

Opposite Carcinogenic Effects of Circadian Clock Gene BMAL1

The circadian clock confers daily rhythmicity on many biochemical and physiological functions and its disruption is associated with increased risks of developing obesity, diabetes, heart disease and cancer. Although, there are studies on the role of Bmal1 in carcinogenesis using germline, conditional or tissue-specific knockouts, it is still not well understood how BMAL1 gene affects cancer-related biological events at the molecular level. We, therefore, took an in vitro approach to understand the contribution of BMAL1 in this molecular mechanism using human breast epithelial cell lines by knocking out BMAL1 gene with CRISPR technology. We preferred epithelial cells over fibroblasts as the most of cancers originate from epithelial cells. After obtaining BMAL1 knockouts by targeting the gene at two different sites from non-tumorigenic MCF10A and invasive tumorigenic MDA-MB-231 cells, we analysed apoptosis and invasion properties of the cell lines as representative events in tumor development. BMAL1 disruption sensitized both cell lines to a bulky-DNA adduct forming agent (cisplatin) and a double-strand break-inducing agent (doxorubicin), while it enhanced the invasive properties of MDA-MB-231 cells. These results show that the disruption of clock genes may have opposing carcinogenic effects.

Frizzled7 Promotes Epithelial-to-mesenchymal Transition and Stemness Via Activating Canonical Wnt/β-catenin Pathway in Gastric Cancer.

Aberrant activation of Wnt signaling is a crucial event in tumor development and metastasis. Wnt signaling is commonly divided into canonical and non-canonical signaling pathways based on whether β-catenin is activated (canonical). The two signaling pathways are initiated by Wnt ligand binding to the surface Frizzled (FZD) receptors, and regulate cancer stem cell self-renewal and epithelial-mesenchymal transition (EMT). Frizzled 7 (FZD7), a member of Frizzled family, promotes cell proliferation and invasiveness in many cancers, suggesting that FZD7 transmitting Wnt signaling is important for driving cancer growth. FZD7 expression has been reported to be up-regulated in human primary gastric cancer tissues. However, the molecular mechanism by which FZD7 promotes gastric cancer(GC) development and progression is not fully understood. Our present study showed that FZD7 was overexpressed in clinical GC samples, and thus was correlated with tumor invasion, lymphatic and organ metastasis, late TNM stages and poor patient survival. The endogenous expression of FZD7 was significantly increased in cancer stem cell-enriched spheres compared with adherent cells. Furthermore, RNA interference-mediated silencing of FZD7 inhibited proliferation, migration and invasion in gastric cancer cells. Moreover, ablation of FZD7 down-regulated EMT and the expression levels of cancer stem cell markers, and these inhibitions were associated with attenuated canonical Wnt/β-catenin signaling. The results suggest that Wnt canonical pathway may contribute to tumorigenesis and metastasis, indicating that FZD7 could be a potential therapeutic target for gastric cancer.

Pathological Bases and Clinical Impact of Intratumor Heterogeneity in Clear Cell Renal Cell Carcinoma.

Intratumor heterogeneity is an inherent event in tumor development that is receiving much attention in the last years since it is responsible for most failures of current targeted therapies. The purpose of this review is to offer clinicians an updated insight of the multiple manifestations of a complex event that impacts significantly patient's life. Clear cell renal cell carcinoma is the most common renal tumor and a paradigmatic example of a heterogeneous neoplasm. Next-generation sequencing has demonstrated that intratumor heterogeneity encompasses genetic, epigenetic, and microenvironmental variability. Currently accepted protocols of tumor sampling seem insufficient in unveiling intratumor heterogeneity with reliability and need to be updated. This variability challenges the precise morphological diagnosis, its molecular characterization, and the selection of optimal personalized therapies in clear cell renal cell carcinoma, a neoplasm traditionally considered chemo- and radio-resistant. We review the state of the art of the different approaches to intratumor heterogeneity in clear cell renal cell carcinomas, from the simple morphology to the most sophisticated massive sequencing tools.