Tumorigenic Process Research Articles

Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

The transient receptor potential ankyrin type 1 (TRPA1) channel belongs to the TRP superfamily of ion channels. TRPA1 is a membrane protein with multiple functions able to respond to noxious stimuli, reactive oxygen species, inflammatory cytokines or pungent substances, and it participates in pain signalling, taste, inflammation and various steps of the tumorigenic process. To date, no reports have addressed the expression and function of TRPA1 in pancreatic ductal adenocarcinoma (PDAC) cells. This work reports the endogenous expression of TRPA1 channels in human pancreatic adenocarcinoma cell lines and provides insights into the function of the TRPA1 protein in the Panc-1 cell line. This study reports that cell lines isolated from PDAC patients had different levels of TRPA1 expression. The channel activity in Panc-1 cells, as assessed with electrophysiological (whole-cell patch clamp) and microfluorimetry methods, showed that non-selective cationic currents were activated by allyl isothiocyanate (AITC) in Panc-1 cells and inhibited by the selective TRPA1 antagonist A-967079. The current elicited by the specific agonist was associated with a robust increase in intracellular Ca2+. Furthermore, siRNA-induced downregulation of TRPA1 enhanced cell migration in the wound healing assay, indicating a possible role of ion channels independent from pore function. Finally, TRPA1 activation changed the cell cycle progression. Taken together, these results support the idea of channel-dependent and independent role for TRPA1 in tumoral processes.

Expression and affinity purification of recombinant mammalian mitochondrial ribosomal small subunit (MRPS) proteins and protein-protein interaction analysis indicate putative role in tumourigenic cellular processes.

Mitochondrial ribosomal small subunit (MRPS) group of proteins is structural constituents of the small subunit of mitoribosomes involved in translation. Recent studies indicate role in tumourigenic process, however, unlike cytosolic ribosomal proteins, knowledge on the role of MRPS proteins in alternate cellular processes is very limited. Mapping protein-protein interactions (PPIs) onto known cellular processes can be a valuable tool to identify novel protein functions. In this study, to identify PPIs of MRPS proteins, we have constructed 31 glutathione-S-transferase (GST)/MRPS fusion clones. GST/MRPS fusion proteins were confirmed by MALDI-TOF analysis. GST pull-downs were performed using eight GST/MRPS proteins (MRPS9, MRPS10, MRPS11, MRPS18B, MRPS31, MRPS33, MRPS38 and MRPS39), GST alone as pull-down control and HEK293 cell lysate as the source for anchor proteins followed by nLC/MS/MS analysis and probable PPIs of eight MRPS proteins were identified. Three PPIs from GST pull-downs and interaction between six MRPS proteins and p53 previously reported in PPI database were validated. The PPI network analysis revealed putative role in cellular processes with implications for tumourigenesis. Gene expression screening of a cancer cell line panel indicated overexpression of MRPS10 and MRPS31 in breast cancer. Co-expression module identification tool analysis of breast cancer gene expression and MRPS10 and MRPS31 PPIs revealed putative role for PPI with acyl-CoA dehydrogenase in fatty acid oxidation process regulated by brain-derived neurotrophic factor signalling pathway.

GFI1-Mediated Upregulation of LINC00675 as a ceRNA Restrains Hepatocellular Carcinoma Metastasis by Sponging miR-942-5p.

Hepatocellular carcinoma (HCC) is a common malignant liver tumor worldwide. Tumor recurrence and metastasis contribute to the bad clinical outcome of HCC patients. Substantial studies have displayed lncRNAs modulate various tumorigenic processes of many cancers. Our current work was aimed to investigate the function of LINC00675 in HCC and to recognize the potential interactions between lncRNAs and microRNAs. GFI1 can exhibit a significant role in the progression of human malignant tumors. Firstly, GFI1 was identified using real-time PCR in HCC tissues and cells. In this work, we indicated GFI1 was remarkably reduced in HCC tissues and cells. Meanwhile, GFI1 specifically interacted with the promoter of LINC00675. Up-regulation of LINC00675 obviously repressed the migration and invasion capacity of SMCC-7721 and QGY-7703 cells in vitro. Moreover, decrease of LINC00675 competitively bound to miR-942-5p that contributed to the miRNA-mediated degradation of GFI1, thus facilitated HCC metastasis. The ceRNA function of LINC00675 in HCC cells was assessed and confirmed using RNA immunoprecipitation assay and RNA pull-down assays in our work. Additionally, we proved overexpression of miR-942-5p promoted HCC progression, which was reversed by the up-regulation of GFI1. In summary, LINC00675 might act as a prognostic marker for HCC, which can inhibit HCC development via regulating miR-942-5p and GFI1.

Genome-Wide Identification and Analysis of the Methylation of lncRNAs and Prognostic Implications in the Glioma.

Glioma is characterized by rapid cell proliferation and extensive infiltration among brain tissues, but the molecular pathology has been still poorly understood. Previous studies found that DNA methylation modifications play a key role in contributing to the pathogenesis of glioma. On the other hand, long noncoding RNAs (lncRNAs) has been discovered to be associated with some key tumorigenic processes of glioma. Moreover, genomic methylation can influence expression and functions of lncRNAs, which contributes to the pathogenesis of many complex diseases. However, to date, no systematic study has been performed to detect the methylation of lncRNAs and its influences in glioma on a genome-wide scale. Here, we selected the methylation data, clinical information, expression of lncRNAs, and DNA methylation regulatory proteins of 537 glioma patients from TCGA and TANRIC databases. Then, we performed a differential analysis of lncRNA expression and methylated regions between low-grade glioma (LGG) and glioblastoma multiform (GBM) subjects, respectively. Next, we further identified and verified potential key lncRNAs contributing the pathogenesis of glioma involved in methylation modifications by an annotation and correlation analysis, respectively. In total, 18 such lncRNAs were identified, and 7 of them have been demonstrated to be functionally linked to the pathogenesis of glioma by previous studies. Finally, by the univariate Cox regression, LASSO regression, clinical correlation, and survival analysis, we found that all these 18 lncRNAs are high-risk factors for clinical prognosis of glioma. In summary, this study provided a strategy to explore the influence of lncRNA methylation on glioma, and our findings will be benefit to improve understanding of its pathogenesis.

Editing and Chemical Modifications on Non-Coding RNAs in Cancer: A New Tale with Clinical Significance.

Currently, for seemingly every type of cancer, dysregulated levels of non-coding RNAs (ncRNAs) are reported and non-coding transcripts are expected to be the next class of diagnostic and therapeutic tools in oncology. Recently, alterations to the ncRNAs transcriptome have emerged as a novel hallmark of cancer. Historically, ncRNAs were characterized mainly as regulators and little attention was paid to the mechanisms that regulate them. The role of modifications, which can control the function of ncRNAs post-transcriptionally, only recently began to emerge. Typically, these modifications can be divided into reversible (i.e., chemical modifications: m5C, hm5C, m6A, m1A, and pseudouridine) and non-reversible (i.e., editing: ADAR dependent, APOBEC dependent and ADAR/APOBEC independent). The first research papers showed that levels of these modifications are altered in cancer and can be part of the tumorigenic process. Hence, the aim of this review paper is to describe the most common regulatory modifications (editing and chemical modifications) of the traditionally considered “non-functional” ncRNAs (i.e., microRNAs, long non-coding RNAs and circular RNAs) in the context of malignant disease. We consider that only by understanding this extra regulatory layer it is possible to translate the knowledge about ncRNAs and their modifications into clinical practice.

TERRA Gene Expression in Gastric Cancer: Role of hTERT.

One of the most important serious malignancies is gastric cancer (GC) with a high mortality globally. In this way, beside the environmental factors, genetic parameter has a remarkable effective fluctuation in GC. Correspondingly, telomeres are nucleoprotein structures measuring the length of telomeres and they have special potential in diagnosis of various types of cancers. Defect protection of the telomeric length initiates the instability of the genome during cancer, including gastric cancer. The most common way of maintaining telomere length is the function of the telomerase enzyme that replicates the TTAGGG to the end of the 3' chromosome. In this review, we want to discuss the alterations of hTERT repression on the modification of TERRA gene expression in conjunction with the importanceof telomere and telomerase in GC. The telomerase enzyme contains two essential components called telomerase reverse transcriptase (hTERT) and RNA telomerase (hTR, hTERC).Deregulation of hTERT plays a key role in the multistage process of tumorigenicity and anticancer drug resistance. The direct relationship betweentelomerase activity and hTERT has led to hTERT to be considered a key target for cancer treatment. Recent results show that telomeres are transcribed intotelomeric repeat-containing RNA (TERRA) in mammalian cells and are long noncoding RNAs (lncRNAs) identified in different tissues. In addition, mostchemotherapy methods have a lot of side effects on normal cells. Telomere and telomerase are useful therapeutic goal. According to the main roles of hTERT in tumorigenesis, growth, migration, and cancer invasion,hTERT and regulatory mechanisms that control the expression of hTERT are attractive therapeutic targets for cancer treatment.

Chronic active EBV infection in refractory enteritis with longitudinal ulcers with a cobblestone appearance: an autopsied case report

BackgroundChronic active Epstein–Barr virus infection (CAEBV) is defined as Epstein–Barr virus (EBV)-positive T/NK cell-related neoplasia, and its major clinical symptom is systemic inflammation presenting as infectious mononucleocytosis, whereas enteritis and diarrhea are minor clinical symptoms. The complex mixture of tumorigenic processes of EBV-positive cells and physical symptoms of systemic inflammatory disease constitutes the varied phenotypes of CAEBV. Herein, we describe a case of CAEBV that was initially diagnosed as Crohn’s disease (CD) based on ileal ulcers and clinical symptoms of enteritis.Case presentationA 19-year-old woman complained of abdominal pain and fever. Blood examination showed normal blood cell counts without atypical lymphocyte but detected modest inflammation, hypoalbuminemia, slight liver dysfunction, and evidence of past EBV infection. The esophagogastroduodenoscopic findings were normal. However, colonoscopy revealed a few small ulcers in the terminal ileum. The jejunum and ileum also exhibited various forms of ulcers, exhibiting a cobblestone appearance, on capsule endoscopy. Based on these clinical findings, she was strongly suspected with CD. In the course of treatment by steroid and biologics for refractory enteritis, skin ulcers appeared about 50 months after her initial hospital visit. Immunohistology of her skin biopsy revealed proliferation of EBV-encoded small RNA (EBER)-positive atypical lymphocytes. We retrospectively assessed her previous ileal ulcer biopsy before treatment and found many EBER-positive lymphocytes. Blood EBV DNA was also positive. Therefore, she was diagnosed with extranodal NK/T-cell lymphoma with CAEBV-related enteritis rather than CD. She was treated with cyclosporine and prednisolone combination therapy for CAEBV-related systemic inflammation and chemotherapy for malignant lymphoma. Unfortunately, her disease continued to progress, leading to multiple organ failure and death at the age of 23 years.ConclusionClinicians need to remember the possibility of CAEBV as a differential diagnosis of refractory enteritis. Enteritis with intestinal ulcer is a rare symptom of CAEBV, and it is impossible to acquire a definitive diagnosis by ulcer morphology only. In cases where the possibility of CAEBV remains, tissue EBVR expression should be checked by in situ hybridization and blood EBV DNA.

CXCL13 Signaling in the Tumor Microenvironment.

Chemokines have emerged as important players in tumorigenic process. An extensive body of literature generated over the last two or three decades strongly implicate abnormally activated or functionally disrupted chemokine signaling in liaising most-if not all-hallmark processes of cancer. It is well-known that chemokine signaling networks within the tumor microenvironment are highly versatile and context-dependent: exert both pro-tumoral and antitumoral activities. The C-X-C motif chemokine ligand 13 (CXCL13), and its cognate receptor CXCR5, represents an emerging example of chemokine signaling axes, which express the ability to modulate tumor growth and progression in either way. Collateral evidence indicate that CXCL13-CXCR5 axis may directly modulate tumor growth by inducing proliferation of cancer cells, as well as promoting invasive phenotypes and preventing their apoptosis. In addition, CXCL13-CXCR5 axis may also indirectly modulate tumor growth by regulating noncancerous cells, particularly the immune cells, within the tumor microenvironment. Here, we review the role of CXCL13, together with CXCR5, in the human tumor microenvironment. We first elaborate their patterns of expression, regulation, and biological functions in normal physiology. We then consider how their aberrant activity, as a result of differential overexpression or co-expression, may directly or indirectly modulate the growth of tumors through effects on both cancerous and noncancerous cells.

Opposite trends of GAS6 and GAS6-AS expressions in breast cancer tissues

Growth arrest-specific gene 6 (GAS6) is a growth factor-like cytokine whose function is related with vitamin K. This protein interacts with receptor tyrosine kinase proteins such as Tyro3, Axl, and TAM Receptor family, therefore affecting the tumorigenic processes via different mechanisms. GAS6-antisense 1 (GAS6-AS1) is a long non-coding RNAs (lncRNAs) that is transcribed from a genomic regions nearby GAS6. This lncRNA is also implicated in the pathobiology of cancer. We intended to judge the role of GAS6 and GAS6-AS1 in the pathogenesis of breast cancer through appraisal of their expression levels in breast cancer tissues and their paired neighboring non-cancerous samples. Expression of GAS6 was up-regulated in breast cancer tissues compared with neighboring tissues (Ratio of Mean Expressions = 2.18, P value = 4.98E-02). On the other hand, expression of GAS6-AS1 was down-regulated in breast tumor tissues compared with controls (Ratio of Mean Expressions = 0.37, P value = 4.26E-03). There were substantial correlations between expression levels GAS6 and GAS6-AS1 in non-cancerous tissues (r = 0.74, P value = 1.47e-13) and cancer tissues (r = 0.85, P value = 2.28e-20). Expression of GAS6-AS was associated with progesterone receptor status (P value = 1.36E-02). However, expressions of this gene and the sense transcript were not linked with any other clinical or demographic variable. Taken together, GAS6 and GAS6-AS1 might partake in the development of breast cancer.

Suppression MGP inhibits tumor proliferation and reverses oxaliplatin resistance in colorectal cancer

Matrix Gla protein (MGP), an extracellular matrix protein, has been widely reported to participate in the tumorigenic process and is abnormally expressed in several tumors. However, the role of MGP in colorectal cancer (CRC) remains unknown. Chemotherapy resistance represents a significant limitation in the treatment of CRC. Here, a comprehensive bioinformatics analysis revealed that MGP, which is overexpressed in CRC, might act as one of the critical genes conferring resistance to oxaliplatin (OXA). Furthermore, we found that MGP overexpression in tumor tissue might be correlated with cancer stage and patient prognosis, consistent with the bioinformatics analysis. The upregulation of MGP may act as an independent risk factor for CRC. The knockdown of MGP or inhibition of MGP expression significantly increased the sensitivity of the CRC cell lines to OXA. Suppression of MGP may reverse OXA resistance by upregulating copper transporter 1 (CTR1) and downregulating ATP7A and ATP7B. When used in combination with OXA, the inhibition of MGP reduced cancer cell proliferation, invasion, and migration and increased cell apoptosis in vitro. Suppression of MGP or OXA treatment alone significantly inhibited tumor growth in the CRC mouse model. Additionally, we found that OXA might promote the antitumor immune response in vivo. In summary, our study is the first to provide evidence that MGP expression confers OXA chemotherapy resistance in CRC and provides novel strategies to overcome chemotherapy resistance in CRC.

A Novel Platform to Test In Vivo Single Gene Dependencies in t(8,21) and t(15,17) AML Confirms Zeb2 as Leukemia Target.

Simple SummaryMouse models are extensively used to study human diseases, including cancer. They are particularly useful to evaluate the role of specific genes in the tumorigenic process. The platform we present allows to effectively induce in vivo silencing of any potential candidate gene in two acute myeloid leukemia mouse models, with the scope of furthering the understanding of this gene’s role in the biology of leukemia.The increased usage of high-throughput technologies in cancer research, including genetic and drug screens, generates large sets of candidate targets that need to be functionally validated for their roles in tumor development. Thus, reliable and robust in vivo model systems are needed to perform reverse genetic experiments. Ideally, these models should allow for a conditional silencing of the target and an unambiguous identification of engineered cancer cells. Here, we present a platform consisting of: (i) t(8;21) and t(15;17) driven acute myeloid leukemia (AML) transgenic mice with constitutive expression of green fluorescent protein (GFP) and inducible expression of Cre recombinase, and (ii) REX, a modified pSico lentiviral vector for inducible shRNA expression and red fluorescent protein (RFP) as a selection marker. In this system, leukemic cells from transgenic mice are transduced with REX, flow sorted, and transplanted into syngeneic hosts. Gene interference is induced in established tumors by tamoxifen treatment. Dual-color cell fluorescence guides the in vivo identification of shRNA interfered AML cells, monitoring engraftment and disease progression. We tested the platform by inducing knockdown of Zeb2, a gene upregulated by AML1-ETO and PML-RARα oncogenes in pre-leukemic hematopoietic stem cell compartment, and observed a significant delay in leukemia onset. This proves the power and utility of the platform and confirms Zeb2 contribution to the pathogenesis of AML.

Identification and Analysis of the Blood lncRNA Signature for Liver Cirrhosis and Hepatocellular Carcinoma.

As one of the most common malignant tumors, hepatocellular carcinoma (HCC) is the fifth major cause of cancer-associated mortality worldwide. In 90% of cases, HCC develops in the context of liver cirrhosis and chronic hepatitis B virus (HBV) infection is an important etiology for cirrhosis and HCC, accounting for 53% of all HCC cases. To understand the underlying mechanisms of the dynamic chain reactions from normal to HBV infection, from HBV infection to liver cirrhosis, from liver cirrhosis to HCC, we analyzed the blood lncRNA expression profiles from 38 healthy control samples, 45 chronic hepatitis B patients, 46 liver cirrhosis patients, and 46 HCC patients. Advanced machine-learning methods including Monte Carlo feature selection, incremental feature selection (IFS), and support vector machine (SVM) were applied to discover the signature associated with HCC progression and construct the prediction model. One hundred seventy-one key HCC progression-associated lncRNAs were identified and their overall accuracy was 0.823 as evaluated with leave-one-out cross validation (LOOCV). The accuracies of the lncRNA signature for healthy control, chronic hepatitis B, liver cirrhosis, and HCC were 0.895, 0.711, 0.870, and 0.826, respectively. The 171-lncRNA signature is not only useful for early detection and intervention of HCC, but also helpful for understanding the multistage tumorigenic processes of HCC.

Exome sequencing identifies ARID2 as a novel tumor suppressor in early-onset sporadic rectal cancer.

Early-onset sporadic rectal cancer (EOSRC) is a unique and predominant colorectal cancer (CRC) subtype in India. In order to understand the tumorigenic process in EOSRC, we performed whole-exome sequencing of 47 microsatellite stable EOSRC samples. Signature 1 was the predominant mutational signature in EOSRC, as previously shown in other CRC exome studies. More importantly, we identified TP53, KRAS, APC, PIK3R1, SMAD4 and ZNF880 as significantly mutated (q < 0.1) and ARID1A and ARID2 as near-significantly mutated (restricted hypothesis testing; q < 0.1) candidate drivers. Unlike the other candidates, the tumorigenic potential of ARID2, encoding a component of the SWI/SNF chromatin remodeling complex, is largely unexplored in CRC. shRNA-mediated ARID2 knockdown performed in different CRC cell lines resulted in significant alterations in transcript levels of cancer-related target genes. More importantly, ARID2 knockdown promoted several tumorigenic features including cell viability, proliferation, ability to override contact inhibition of growth, and migration besides significantly increasing tumor formation ability in nude mice. The observed gain in tumorigenic features was rescued upon ectopic expression of wild type but not mutant ARID2. Analyses of the TCGA pan-cancer dataset revealed several modes of ARID2 inactivation and of the CRC dataset revealed poorer survival in patients with ARID2 alterations. We therefore propose ARID2 as a novel tumor suppressor in CRC.

Abstract IA41: Novel Approaches for Accessing Molecular Heterogeneity

Abstract Ovarian cancer is the fifth leading cause of cancer-related deaths in women and is the deadliest gynecological malignancy in the United States. The standard treatment of ovarian cancer is based on debulking surgery followed by platinum- and taxane-based chemotherapy, and has remained the same over the past three decades. Over those years, molecular targeted and combination therapies have been developed and clinically approved, however the overall survival rate has not improved significantly due to chemo-resistance. Further, the majority of patients experience recurrence of treatment-resistant tumors. The genomic diversity within a tumor and the varying cell types within its microenvironment has placed significant importance on heterogeneity and its clinical implications. Intra-tumor heterogeneity has often been blamed for treatment failure in ovarian carcinoma. Consequently, intra-tumor heterogeneity is a key factor driving drug resistance, therapeutic failure, and poor outcomes and poses a significant challenge to personalized cancer medicine. Intra-tumor heterogeneity is a hallmark of cancer where the molecular and cellular interactions within the tumor microenvironment can dictate a cancer’s fate. Molecular profiling of bulk tissue specimens using methods such as whole-transcriptome sequencing are limited in their ability to resolve fine grain molecular signatures and hinder our utility to dissect the underlying biology of individual tumors. Although informatics approaches are available that attempt to disentangle tissue heterogeneity from bulk tumor data, emerging spatial whole transcriptome sequencing technologies allow a more precise delineation of cellular and molecular substructure in a comprehensive unbiased approach. Spatial Transcriptomics (ST) enables high-throughput whole transcriptomic sequencing within a single intact tissue by using a glass slide arrayed with barcoded cDNA primers at a resolution of 100um (3-30 cells). This workflow requires no tissue dissociation keeping fragile cell types intact. Resulting data from this workflow is overlaid on the tissue, displayed as a “cluster reference map”, providing comprehensive unbiased transcriptional substructure and unique possibilities for subsequent in situ analysis. The overall goal of this study is to utilize ST to unveil the unexplored landscape of intra-tumor heterogeneity in ovarian cancer and determine its translational relevance. Here, we applied ST to profile gene expression in fresh frozen OCT embedded sections from nine high grade ovarian patients. Three serial 5-micron frozen sections were placed on proprietary 10x Genomics Spatial Transcriptomics (ST) slides and processed using manufacturer specifications. Libraries were sequenced on the Illumina NovaSeq 6000 system and data was processed using 10X Genomics analytical tools. Next, we aggregated the transcriptional profiles of serial sections from each case increasing our power to cluster similar regions and identify differentially expressed genes within these tissues. Using serial sections of solid tumors from each subject we were not only able to profile each section at 100um resolution but also spatially resolve gene expression signatures and cluster regions of tissue based on these signatures. This ST workflow reliably quantitated an average spatial distribution of 19,285 genes per section in our ovarian cancer cohort. Interestingly, within our cohort we have extreme outliers that include primary tumors that did not have any response to standard adjuvant chemotherapy, paclitaxel and carboplatin, and patients that sustained a durable response to standard adjuvant chemotherapy and diagnosed as disease free ≥3 years and then presented with recurrent disease. We identified tumor heterogeneity as unique spatially-resolved gene expression clusters across each tissue section defined by individual gene sets associated with tumorigenic molecular processes, immune cell quantity and localization. These approaches highlight the power of spatial whole-transcriptomic sequencing in solid tumor studies to help unravel the complexity of heterogeneous cancers and provide a comprehensive characterization of transcriptional substructure within a single tissue section. Citation Format: Lee D. Gibbs, Lynda Roman, Michelle Webb, Rania Bassiouni, Stephen R Williams, Neil I. Weisenfeld, Nigel F. Delaney, Yifeng Yin, Solomon Rotimi, Jennifer Chew, Meghan Frey, Jing Qian, Heather Miller, Laila Murderspach, Diane Da Silva, Troy McEachron, David W. Craig, John D. Carpten. Novel Approaches for Accessing Molecular Heterogeneity [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr IA41.

LDL, HDL and endocrine-related cancer: From pathogenic mechanisms to therapies.

Cholesterol is essential for a variety of functions in endocrine-related cells, including hormone and steroid production. We have reviewed the progress to date in research on the role of the main cholesterol-containing lipoproteins; low-density lipoprotein (LDL) and high-density lipoprotein (HDL), and their impact on intracellular cholesterol homeostasis and carcinogenic pathways in endocrine-related cancers. Neither LDL-cholesterol (LDL-C) nor HDL-cholesterol (HDL-C) was consistently associated with endocrine-related cancer risk. However, preclinical studies showed that LDL receptor plays a critical role in endocrine-related tumor cells, mainly by enhancing circulating LDL-C uptake and modulating tumorigenic signaling pathways. Although scavenger receptor type BI-mediated uptake of HDL could enhance cell proliferation in breast, prostate, and ovarian cancer, these effects may be counteracted by the antioxidant and anti-inflammatory properties of HDL. Moreover, 27-hydroxycholesterol a metabolite of cholesterol promotes tumorigenic processes in breast and epithelial thyroid cancer. Furthermore, statins have been reported to reduce the incidence of breast, prostate, pancreatic, and ovarian cancer in large clinical trials, in part because of their ability to lower cholesterol synthesis. Overall, cholesterol homeostasis deregulation in endocrine-related cancers offers new therapeutic opportunities, but more mechanistic studies are needed to translate the preclinical findings into clinical therapies.

HIC1 (Hypermethylated in Cancer 1) modulates the contractile activity of prostate stromal fibroblasts and directly regulates CXCL12 expression.

HIC1 (Hypermethylated In Cancer 1) a tumor suppressor gene located at 17p13.3, is frequently deleted or epigenetically silenced in many human tumors. HIC1 encodes a transcriptional repressor involved in various aspects of the DNA damage response and in complex regulatory loops with P53 and SIRT1. HIC1 expression in normal prostate tissues has not yet been investigated in detail. Here, we demonstrated by immunohistochemistry that detectable HIC1 expression is restricted to the stroma of both normal and tumor prostate tissues. By RT-qPCR, we showed that HIC1 is poorly expressed in all tested prostate epithelial lineage cell types: primary (PrEC), immortalized (RWPE1) or transformed androgen-dependent (LnCAP) or androgen-independent (PC3 and DU145) prostate epithelial cells. By contrast, HIC1 is strongly expressed in primary PrSMC and immortalized (WMPY-1) prostate myofibroblastic cells. HIC1 depletion in WPMY-1 cells induced decreases in α-SMA expression and contractile capability. In addition to SLUG, we identified stromal cell-derived factor 1/C-X-C motif chemokine 12 (SDF1/CXCL12) as a new HIC1 direct target-gene. Thus, our results identify HIC1 as a tumor suppressor gene which is poorly expressed in the epithelial cells targeted by the tumorigenic process. HIC1 is expressed in stromal myofibroblasts and regulates CXCL12/SDF1 expression, thereby highlighting a complex interplay mediating the tumor promoting activity of the tumor microenvironment. Our studies provide new insights into the role of HIC1 in normal prostatic epithelial-stromal interactions through direct repression of CXCL12 and new mechanistic clues on how its loss of function through promoter hypermethylation during aging could contribute to prostatic tumors.

Evidence for functional and regulatory cross-talk between Wnt/β-catenin signalling and Mre11-Rad50-Nbs1 complex in the repair of cisplatin-induced DNA cross-links.

The canonical Wnt/β-catenin signalling pathway plays a crucial role in a variety of functions including cell proliferation and differentiation, tumorigenic processes and radioresistance in cancer cells. The Mre11–Rad50–Nbs1 (MRN) complex has a pivotal role in sensing and repairing DNA damage. However, it remains unclear whether a connection exists between Wnt/β-catenin signalling and the MRN complex in the repair of cisplatin-induced DNA interstrand cross-links (ICLs). Here, we report that (1) cisplatin exposure results in a significant increase in the levels of MRN complex subunits in human tumour cells; (2) cisplatin treatment stimulates Wnt/β-catenin signalling through increased β-catenin expression; (3) the functional perturbation of Wnt/β-catenin signalling results in aberrant cell cycle dynamics and the activation of DNA damage response and apoptosis; (4) a treatment with CHIR99021, a potent and selective GSK3β inhibitor, augments cisplatin-induced cell death in cancer cells. On the other hand, inactivation of the Wnt/β-catenin signalling with FH535 promotes cell survival. Consistently, the staining pattern of γH2AX-foci is significantly reduced in the cells exposed simultaneously to cisplatin and FH535; and (5) inhibition of Wnt/β-catenin signalling impedes cisplatin-induced phosphorylation of Chk1, abrogates the G2/M phase arrest and impairs recombination-based DNA repair. Our data further show that Wnt signalling positively regulates the expression of β-catenin, Mre11 and FANCD2 at early time points, but declining thereafter due to negative feedback regulation. These results support a model wherein Wnt/β-catenin signalling and MRN complex crosstalk during DNA ICL repair, thereby playing an important role in the maintenance of genome stability.

Cytotoxic effects of Ridolfia segetum (L.) Moris phytoproducts in cancer cells

Ethnopharmacological relevanceThe past few years have witnessed an increasing interest in essential oils (EOs) as potential therapeutic agents against a wide variety of pathologies, including cancer. EOs extracted from Ridolfia segetum (L.) Moris (R. segetum) are a clear example of a phytoproduct with therapeutic applications, as it is widely used in traditional medicine due to its antioxidant and anti-inflammatory properties, and these properties were already validated by previous studies. Although, it is well established that inflammation is a key hallmark of cancer, with a key role promoting tumorigenesis, and being chronic inflammation often associated with tumorigenic processes, there are no previous studies regarding the assessment of the antitumoural potential of R. segetum EOs. Aim of the studyThe present study intends to be the first to evaluate the antitumoural proprieties of R. segetum EO phytoproducts in cancer cell models. Materials and methodsFor this, R. segetum EOs were extracted from plants collected at either flowering (RS_Fl) or fruiting (RS_Fr) stage. The impact on proliferation and viability of treatment with R. segetum EO extracts was assessed using in vitro 2D and 3D models. ResultsBoth R. segetum EOs presented effective antiproliferative/viability effects, evidence noted by low IC50 values in 2D models, and significant reduction of spheroid size in 3D in vitro models. Mechanistically, treatment with R. segetum EOs was associated with an altered G1 (associated with p21 stabilisation), and subsequent induction of apoptosis. ConclusionsOverall, these results indicate that R. segetum EOs have potential as suitable antitumoural therapeutic agents.

Notch3 is involved in the proliferation of renal cancer cells via regulation of cell cycle progression and HIF-2α.

Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Although deregulation of the Notch signaling pathway is common in RCC and is involved in the tumorigenic process, the exact role of Notch3 and its underlying molecular mechanism in RCC, particularly in hypoxia, remain unknown. In the present study, RO4929097, a Notch3 inhibitor, was used to alter NICD3 expression. A Cell Counting Kit-8 assay, EdU incorporation assay, colony formation assay, flow cytometry and western blot analysis were used to investigate the effects of altered NICD3 expression on cell proliferation, cell cycle progression and HIF-2α protein expression. The results of western blot analysis showed that RO4929097 dose-dependently decreased the expression of Notch3 intracellular domain (NICD3) in 786-O and ACHN cells, which originate from clear cell RCC (ccRCC). The results of the Cell Counting Kit-8, EdU incorporation and colony formation assays demonstrated that downregulation of NICD3 significantly suppressed cell proliferation in both normoxia and hypoxia. In addition, flow cytometry and western blot analysis demonstrated that hypoxia (2% O2) promoted cell cycle progression in ccRCC cells with the increased expression of G1-S transition-associated proteins, namely cyclin-dependent kinase (CDK)4 and cyclin D1, while downregulation of NICD3 exerted negative effects on cell cycle progression, and the expression levels of CDK4 and cyclin D1. Furthermore, western blot analysis revealed that 2% O2-induced upregulated hypoxia-inducible factor-2α (HIF-2α) expression decreased following downregulation of NICD3 in 786-O and ACHN cells. Following transfection of the vector containing the NICD3 coding sequence, HIF-2α, CDK4, cyclin D1 and proliferating cell nuclear antigen expression, that were inhibited by RO4929097 in hypoxia, were rescued. Collectively, the results of the present study suggest that Notch3 is closely associated with the cell proliferation of ccRCC cells by regulating the cell cycle and HIF-2α.

Hsa-miR-27a-3p and epidermal growth factor receptor expression analysis in glioblastoma FFPE samples.

Glioblastoma multiforme (GBM) is the most invasive type of glial tumors. MicroRNAs as the small, noncoding RNAs have been revealed that play an important role in tumorigenic processes. So, they may be used as potential biomarkers for detection and prognosis of cancers at the early stages. In addition, they can be applied as therapeutic targets. In the present study, the expression levels of hsa-miR-27a-3p and EGFR were investigated in GBM. Real-time RT-PCR was applied to evaluate hsa-miR-27a-3p and EGFR expressions in the formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from 50GBM and 50 normal people. The expression level of hsa-miR-27a-3p and EGFR was significantly different between cases and controls. Positive association was found between gene expressions and immunohistochemistry markers, such as Ki67 and glial fibrillary acidic protein, except for IDH1 status. We showed the association of hsa-miR-27a-3p and EGFR with GBM and it can be concluded that they have a promising potential to use as primary cancer biomarkers.