Malignant Transformation Of Normal Cells Research Articles

Association Study of Pleural Mesothelioma and Oncogenic Simian Virus 40 in the Crocidolite-Contaminated Area of Dayao County, Yunnan Province, Southwest China.

Background: In Dayao County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, Southwest China, 5% of the surface is scattered with blue asbestos, which has a high incidence of pleural mesothelioma (PMe). Simian virus 40 (SV40) is a small circular double-stranded DNA polyomavirus that can cause malignant transformation of normal cells of various human and animal tissue types and promote tumor growth. In this study, we investigate whether oncogenic SV40 is associated with the occurrence of PMe in the crocidolite-contaminated area of Dayao County, Yunnan Province, Southwest China. Methods: Tumor tissues from 51 patients with PMe (40 of whom had a history of asbestos exposure) and pleural tissues from 12 non-PMe patients (including diseases such as pulmonary maculopathy and pulmonary tuberculosis) were collected. Three pairs of low-contamination risk primers (SVINT, SVfor2, and SVTA1) were used to detect the gene fragment of SV40 large T antigen (T-Ag) by polymerase chain reaction (PCR). The presence of SV40 T-Ag in PMe tumor tissues and PMe cell lines was detected by Western blotting and immunohistochemical staining with SV40-related antibodies (PAb 101 and PAb 416). Results: PCR, Western blotting, and immunohistochemical staining results showed that the Met5A cell line was positive for SV40 and contained the SV40 T-Ag gene and protein. In contrast, the various PMe cell lines NCI-H28, NCI-H2052, and NCI-H2452 were negative for SV40. PCR was negative for all three sets of low-contamination risk primers in 12 non-PMe tissues and 51 PMe tissues. SV40 T-Ag was not detected in 12 non-PMe tissues or 51 PMe tissues by immunohistochemical staining. Conclusion: Our data suggest that the occurrence of PMe in the crocidolite-contaminated area of Yunnan Province may not be related to SV40 infection and that crocidolite exposure may be the main cause of PMe. The Clinical Trial Registration number: 2020-YXLL20.

MiR-374a/b-5p Suppresses Cell Growth in Papillary Thyroid Carcinoma Through Blocking Exosomal ANXA1-Induced Macrophage M2 Polarization.

Papillary thyroid carcinoma (PTC), comprising 85% of all thyroid cancers, is an epithelial malignancy. The potential for malignant transformation in normal cells by thyroid cancer cells via exosomal Annexin A1 (ANXA1) delivery is investigated in this study. Our aim is to determine the impact of PTC cells on macrophage polarization through exosomal ANXA1 secretion and its implications for tumor progression.Exosomes in PTC cells were examined using transmission electron microscopy, exosome labeling, and nanoparticle tracking analysis. Real-time quantitative polymerase chain reaction was employed to quantify gene expression levels. Protein levels were determined through Western blot analysis. The interplay between genes was assessed using luciferase reporter and RNA pull-down assays. Functional experiments were conducted to investigate PTC cell proliferation and apoptosis.Our findings reveal that ANXA1 promotes PTC cell proliferation and inhibits apoptosis. Exosomes derived from PTC cells were found to promote macrophage M2 polarization. ANXA1 stimulates M2 polarization through the activation of the PI3K/AKT pathway. MicroRNA-374a-5p (miR-374a-5p) and microRNA-374b-5p (miR-374b-5p) were identified as inhibitors of ANXA1 expression and PI3K/AKT pathway activity, thereby inhibiting macrophage M2 polarization. Furthermore, miR-374a-5p and miR-374b-5p were observed to suppress PTC cell proliferation through their regulatory action on ANXA1.Our study suggests that miR-374a/b-5p inhibits PTC cell growth by blocking the macrophage M2 polarization induced by exosomal ANXA1.

HPV16 E6/E7-mediated regulation of PiwiL1 expression induces tumorigenesis in cervical cancer cells.

PiwiL1 has been reported to be over-expressed in many cancers. However, the molecular mechanism by which these proteins contribute to tumorigenesis and their regulation in cancer cells is still unclear. We intend to understand the role of PiwiL1 in tumorigenesis and also its regulation in cervical cells. We studied the effect of loss of PiwiL1 function on tumor properties of cervical cancer cells in vitro and in vivo. Also we have looked into the effect of PiwiL1 overexpression in the malignant transformation of normal cells both in vitro and in vivo. Further RNA-seq and RIP-seq analyses were done to get insight of the direct and indirect targets of PiwiL1 in the cervical cancer cells. Here, we report that PiwiL1 is not only over-expressed, but also play a major role in tumor induction and progression. Abolition of PiwiL1 in CaSki cells led to a decrease in the tumor-associated properties, whereas, its upregulation conferred malignant transformation of normal HaCaT cells. Our study delineates a new link between HPV oncogenes, E6 and E7 with PiwiL1. p53 and E2F1 directly bind and differentially regulate PiwiL1 promoter in a context-dependant manner. Further, RNA-seq together with RIP-RNA-seq suggested a strong and direct role for PiwiL1 in promoting metastasis in cervical cancer cells. Our study demonstrates that PiwiL1 act as an oncogene in cervical cancer by inducing tumor-associated properties and EMT pathway. The finding that HPV oncogenes, E6/E7 can positively regulate PiwiL1 suggests a possible mechanism behind HPV-mediated tumorigenesis in cervical cancer.

Overview of Cancer Metabolism and Signaling Transduction.

Despite the remarkable progress in cancer treatment up to now, we are still far from conquering the disease. The most substantial change after the malignant transformation of normal cells into cancer cells is the alteration in their metabolism. Cancer cells reprogram their metabolism to support the elevated energy demand as well as the acquisition and maintenance of their malignancy, even in nutrient-poor environments. The metabolic alterations, even under aerobic conditions, such as the upregulation of the glucose uptake and glycolysis (the Warburg effect), increase the ROS (reactive oxygen species) and glutamine dependence, which are the prominent features of cancer metabolism. Among these metabolic alterations, high glutamine dependency has attracted serious attention in the cancer research community. In addition, the oncogenic signaling pathways of the well-known important genetic mutations play important regulatory roles, either directly or indirectly, in the central carbon metabolism. The identification of the convergent metabolic phenotypes is crucial to the targeting of cancer cells. In this review, we investigate the relationship between cancer metabolism and the signal transduction pathways, and we highlight the recent developments in anti-cancer therapy that target metabolism.

MicroRNA-613 Enhances Nasopharyngeal Carcinoma Cell Radiosensitivity via the DNA Methyltransferase 3B/Tissue Inhibitor of Matrix Metalloproteinase-3/Signal Transducer and Activator of Transcription-1/Forkhead Box O-1 Axis.

Nasopharyngeal carcinoma (NPC) is a common malignancy of the nasopharynx, and radioresistant represents the main obstacle in NPC treatment. Malignant transformation of normal cells is driven by genetic and epigenetic changes, which are primarily manifested as changes in miRNA levels and DNA methylation status. microRNA (miR)-613 plays an inhibitory role in several types of cancer. Herein, the current study sought to explore the roles of miR-613 in NPC cell radiosensitivity. miR-613 expression patterns in NPC tissues were detected, and its correlation with clinical indexes was analyzed. NP-69 and C666-1 cell lines were selected for cellular experimentation. Radioresistant cell line C666-1R was obtained by fractionated radiation. Cell viability, survival fraction, and apoptosis were detected by CCK-8, colony formation assay, and flow cytometry. The binding relation between miR-613 and DNMT3B was verified by dual-luciferase and RIP assays. miR-613 was lowly expressed in NPC tissues and cells, with lower expression levels in C666-1R than C666-1, and further correlated with lymph node metastasis, tumor size, and tumor metastasis. miR-613 overexpression reduced C666-1R cell viability and survival fraction and increased apoptosis, while C666-1 cells with silencing miR-613 presented the opposite trends. miR-613 targeted DNMT3B. miR-613 and DNMT3B overexpression led to enhanced C666-1R cell viability and survival fraction and decreased apoptosis. miR-613 reduced TIMP3 methylation and elevated TIMP3 protein level by inhibiting DNMT3B. miR-613 enhanced NPC radiosensitivity by inhibiting the DNMT3B/TIMP3/STAT1/FOXO1 pathway. Collectively, miR-613 inhibited DNMT3B, reduced TIMP3 methylation, and increased TIMP3 protein level, thus inhibiting the STAT1/FOXO1 pathway and enhancing the radiosensitivity of NPC cells.

Contribution of Tumor-Derived Extracellular Vesicles to Malignant Transformation of Normal Cells.

Tumor-cell-derived extracellular vesicles (EVs) are known to carry biologically active molecules of parental cells, which can actively modulate the tumor microenvironment. EVs produced by tumor cells play significant roles in the development and maintenance of tumor growth, metastasis, immune escape, and other important processes. However, the ability of EVs to induce the transformation of normal cells has hardly been investigated. This review discusses studies that describe the ability of tumor-cell-derived EVs to alter the metabolism and morphology of normal cells, causing changes associated with malignant transformation. Additionally, the horizontal transfer of oncogenes through EVs of tumor cells and the induction of epigenetic changes in normal cells, which leads to genomic instability and subsequent oncogenic transformation of normal cells, are also discussed.

The Role of DNA Methylation and DNA Methyltransferases in Cancer.

The malignant transformation of normal cells is driven by both genetic and epigenetic changes. With the advent of next-generation sequencing and large-scale international consortia, it is now possible to profile the genomes and epigenomes of thousands of primary tumors from nearly every cancer type. These studies clearly demonstrate that the dynamic regulation of DNA methylation is a critical epigenetic mechanism of cancer initiation, maintenance, and progression. Proper control of DNA methylation is not only crucial for regulating gene transcription and tissue-specific cellular functions, but its broader consequences include maintaining the integrity of the genome and modulating the immune response. Here, we describe the aberrant DNA methylation changes in human cancers and how they contribute to the disease phenotypes. Aside from CpG island promoter DNA hypermethylation-based gene silencing, human cancers also display gene body DNA hypomethylation that is also associated with downregulated gene expression. In addition, the implementation of whole genome bisulfite sequencing (WGBS) has unveiled DNA hypomethylation of large blocks of the genome, known as partially methylated domains (PMDs), as well as cancer-specific DNA methylation aberrancies at enhancers and super-enhancers. Integrating WGBS and DNA methylation array data with mutation, copy number, and gene expression data has allowed for the identification of novel tumor suppressor genes and candidate driver genes of the disease state. Finally, we highlight potential clinical implications of these changes in the context of prognostic and diagnostic biomarkers, as well as therapeutic targets. Mounting evidence shows that DNA methylation data are effective and highly-sensitive disease classifiers, not only from analyses of the primary tumor but also from tumor-derived, cell free DNA (cfDNA) in blood of cancer patients. These findings highlight the power of DNA methylation aberrancies in providing efficacious biomarkers for clinical utility in improving patient diagnostics and their reversal using DNA methylation inhibitors in cancer treatment may be key in surveillance, treatment, and quality of life for cancer patients.

Identification of a macrophage activation threshold associated with malignant transformation and the sensing of candidate “danger signals”

Abstract Cancer immunosurveillance is based on the recognition of changes brought by malignant transformation of normal cells, leading to their elimination, immunoediting, or escape. The ability of immune cells to sense changes associated with malignant transformation as early as possible is likely to be important for the successful outcome of cancer immunosurveillance. In this process, the immune system faces a trade-off between elimination of cells harboring premalignant or malignant changes, and autoimmune pathologies. We hypothesized that the immune system has therefore evolved a threshold for distinguishing normal from abnormal cells, perhaps similar to a pathogen recognition threshold at which macrophages distinguish non-dangerous from dangerous microbes. We co-cultured human macrophages with a unique set of genetically related human cell lines that recapitulate breast cancer development: MCF10A (immortalized); MCFneoT (hyperplasia); MCFT1 (atypical hyperplasia); MCFCA1 (invasive cancer). Using cytokines-based assays, we found that the threshold for macrophage activation was between MCFNeoT and MCFT1, with macrophages co-cultured with the atypical hyperplasia and invasive cancer cell lines showing an inflammatory cytokine response. This response was accompanied by an increase in macrophage migration, phagocytosis and capacity to infiltrate MCFT1 spheroids. We are currently validating candidates for “danger signals” that will be shared by MCFT1 and MCFCA1 but absent or expressed at low level in the early state of premalignancy. Using proteomic and transcriptomic approaches, we identified 13 surface or secreted molecules corresponding to this definition, some of which are well-known tumor-associated antigens.

Cooperation between NRF2-mediated transcription and MDIG-dependent epigenetic modifications in arsenic-induced carcinogenesis and cancer stem cells.

Environmental exposure to arsenic, a well-established carcinogen linked to a number of human cancers, is a public health concern in many areas of the world. Despite extensive studies on the molecular mechanisms of arsenic-induced carcinogenesis, how initial cellular responses, such as activation of stress kinases and the generation of reactive oxygen species, converge to affect the transcriptional and/or epigenetic reprogramming required for the malignant transformation of normal cells or normal stem cells remains to be elucidated. In this review, we discuss some recent discoveries showing how the transcription factor NRF2 and an epigenetic regulator, MDIG, contribute to the arsenic-induced generation of cancer stem-like cells (CSCs) as determined by applying CRISPR-Cas9 gene editing and chromosome immunoprecipitation followed by DNA sequencing (ChIP-seq).

CRL4ADTL degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation

Genomic instability induced by DNA damage and improper DNA damage repair is one of the main causes of malignant transformation and tumorigenesis. DNA double strand breaks (DSBs) are the most detrimental form of DNA damage, and nonhomologous end-joining (NHEJ) mechanisms play dominant and priority roles in initiating DSB repair. A well-studied oncogene, the ubiquitin ligase Cullin 4A (CUL4A), is reported to be recruited to DSB sites in genomic DNA, but whether it regulates NHEJ mechanisms of DSB repair is unclear. Here, we discovered that the CUL4A-DTL ligase complex targeted the DNA-PKcs protein in the NHEJ repair pathway for nuclear degradation. Overexpression of either CUL4A or DTL reduced NHEJ repair efficiency and subsequently increased the accumulation of DSBs. Moreover, we demonstrated that overexpression of either CUL4A or DTL in normal cells led to genomic instability and malignant proliferation. Consistent with the in vitro findings, in human precancerous lesions, CUL4A expression gradually increased with increasing malignant tendency and was negatively correlated with DNA-PKcs and positively correlated with γ-H2AX expression. Collectively, this study provided strong evidence that the CUL4A-DTL axis increases genomic instability and enhances the subsequent malignant transformation of normal cells by inhibiting NHEJ repair. These results also suggested that CUL4A may be a prognostic marker of precancerous lesions and a potential therapeutic target in cancer.

Integrated molecular characterisation of the MAPK pathways in human cancers reveals pharmacologically vulnerable mutations and gene dependencies

The mitogen-activated protein kinase (MAPK) pathways are crucial regulators of the cellular processes that fuel the malignant transformation of normal cells. The molecular aberrations which lead to cancer involve mutations in, and transcription variations of, various MAPK pathway genes. Here, we examine the genome sequences of 40,848 patient-derived tumours representing 101 distinct human cancers to identify cancer-associated mutations in MAPK signalling pathway genes. We show that patients with tumours that have mutations within genes of the ERK-1/2 pathway, the p38 pathways, or multiple MAPK pathway modules, tend to have worse disease outcomes than patients with tumours that have no mutations within the MAPK pathways genes. Furthermore, by integrating information extracted from various large-scale molecular datasets, we expose the relationship between the fitness of cancer cells after CRISPR mediated gene knockout of MAPK pathway genes, and their dose-responses to MAPK pathway inhibitors. Besides providing new insights into MAPK pathways, we unearth vulnerabilities in specific pathway genes that are reflected in the re sponses of cancer cells to MAPK targeting drugs: a revelation with great potential for guiding the development of innovative therapies.

Evolutionary genomics of mammalian lung cancer genes reveals signatures of positive selection in APC, RB1 and TP53

Lung cancer is the type of cancer causing most deaths in humans, with 234,030 new cases of lung cancer diagnosed in the United States in 2018. Recently, Tumor suppressor genes (TSGs) or the control of its pathway became promising drug targets for cancer therapy. A diverse group of TSGs is involved in progression and metastasis of lung cancer. Here, we surveyed nine highly significant mutated genes in 20 mammalian genomes to assess signatures of adaptive evolution using maximum likelihood approaches. We found that three genes (APC, RB1, and TP53) are under strong positive selection, influencing amino acids located in functionally important protein domains, such as three sites in APC found in the APC_N_CC domain, which is responsible for the binding to beta-catenin armadillo repeats that regulate beta-catenin level (beta-catenin is a transcription factor and its misregulation lead to malignant transformation of normal cells). Such sites substitutions mostly increase the stability of the domain. Moreover, substitution of some other sites found in important motifs, such as codon 47 (proline-directed kinase motif) in TP53, modify the phosphorylation activity of TP53 playing a key role in cancer risk. Our findings will open recommendation to drug targeting sites and will foster further research to understand better these proteins function.

Abstract 36: Synthetic lethality across normal tissues is strongly associated with cancer risk, onset, and tumor suppressor specificity

Abstract The tissue-specificity of cancer and cancer risk is a fundamental open research question. Beyond advancing our understanding of carcinogenesis, elucidating the factors underlying cancer risk may also contribute to cancer prevention. Recent studies have shown that the variation in tissue cancer risk can be explained by the number of tissue stem cell divisions occurring during the lifetime and by abnormal levels of DNA methylation. While cancer risk is likely not determined by a single factor, no other factor has been reported since to account for this fundamental variation. Here we show that cancer synthetic lethality (SL) is another strong contributor of cancer risk in human tissues. SL is a well-known type of genetic interaction where cell death occurs under the combined inactivation of two paired SL genes but not either of them alone. Targeting SLs has been recognized as a highly valuable approach for cancer treatment. We hypothesized that since down-regulated cancer SL (cSL) gene pairs reduce the viability of cancer cells, they may impede the malignant transformation of normal cells, thus modulating cancer risk. Utilizing several recently published large-scale cancer SL networks, we systematically quantified the cancer SL load (defined as the fraction of down-regulated cancer SL gene pairs) in numerous normal and cancer tissues from the TCGA and GTEx datasets. Our key findings are: 1. The cSL loads are lower in cancers vs their matched normal tissues. Furthermore, we find that the cSL load decreases progressively as cancers develop from normal tissues through multiple stages of pre-malignant lesions. These results testify that high cSL load is detrimental to cancer cells, acting as a barrier to cancer development. That is, as normal cells undergo malignant transformation, they need to reactivate at least some of the down-regulated cSL genes for the emerging cancer cells to survive. 2. In accordance with these observations, we find that cSL load in normal tissues is strongly inversely correlated with their lifetime cancer risk. 3. We find that cSL load is the first identified predictor of cancer onset time across different normal tissues - higher cSL load in the younger population is associated with later onset of cancers in that tissue. 4. cSL are important contributors of the tissue/cancer-type specificity of numerous tumor suppressor genes (like BRCA1) - that is, the activity state of cSL partners of quite a few tumor suppressor genes predicts the specific tissues in which they are known to drive cancer. Taken together, these results show that synthetic lethality load in normal tissues is a novel important biological contributor of cancer risk in humans. While synthetic lethality has been attracting tremendous attention as a way to identify cancer vulnerabilities and target them, this is the first time that its role in mediating cancer development is uncovered. Citation Format: Nishanth Ulhas Nair, Kuoyuan Cheng, Joo Sang Lee, Eytan Ruppin. Synthetic lethality across normal tissues is strongly associated with cancer risk, onset, and tumor suppressor specificity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 36.

Cancerous Phenotypes Exhibited by NIH/3T3 Fibroblasts Overexpressing BST‐2

Oncogenesis is a multistep process initiated by the accumulation of genetic and/or epigenetic changes that culminate in the transformation of normal cells into cancer cells. The genetic and/or epigenetic changes result in dysregulated gene expression signature that may involve key cancer driving genes such as Bone Marrow Stromal Antigen 2 (BST‐2). In this study, we sought to test the hypothesis that BST‐2, a type II transmembrane protein regulates early events of oncogenesis. BST‐2 is a multifaceted protein with both antiviral and proviral functions and has been implicated in promoting aggressive cancer cell behavior such as migration and invasion. Meta‐analysis of breast cancer datasets from The Cancer Genome Atlas (TCGA) show a statistically significant correlation between elevated BST‐2 expression and poor survival. In addition to its role in breast cancer progression, elevated BST‐2 expression correlates with poor prognosis in endometrial, brain, lung, and oral cavity cancers, further implicating elevated BST‐2 expression in cancer progression. However, the role of BST‐2 in regulating early oncogenic events is not well understood. Our approach consisted of stably overexpressing exogenous human BST‐2 in NIH/3T3 mouse embryonic fibroblasts that express very low endogenous mouse BST‐2 to generate 3T3BST‐2 cells. The control cells were stably transfected with pCDNA3.1 to generate 3T3pCDNA cells. The cells were tested for phenotypes associated with early oncogenic events under two‐dimensional (2D) and three‐dimensional (3D) culture conditions. In a confluent 2D monolayer, 3T3BST‐2 cells compared to 3T3pCDNA cells resisted contact inhibition and had enhanced growth and survival that is associated with increased phosphorylated protein kinase AKT (T308), increased phosphorylated mitogen‐activated protein kinase (ERK1/2), and decreased pro‐apoptotic factor Bak. In a 3D spheroids model, 3T3BST‐2 spheroids were larger in area and had higher ATP content, an indication of anchorage‐independent survival. Suspended as single cells in a gel, 3T3BST‐2 cells also had enhanced survival, especially when arginylglycylaspartic acid (RGD) adhesive peptide motif was present. Suppressed contact inhibition and enhanced anchorage‐independent survival suggests that overexpression of BST‐2 contributes to malignant transformation of normal cells. Discerning the mechanisms of BST‐2 mediated oncogenesis will expand our understanding of events that transforms a normal cell into an unwanted cancerous cell, which may potentially form the foundation for the development of BST‐2‐targeted cancer therapies.Support or Funding Information1. Molecular and Cellular Pharmacology PhD program 2. CIE’s IMSD‐MERGE scholars program 3. Scholars in Biomedical Sciences program (SBMS) 4. Stony Brook University Cancer Center

MicroRNA expression in melanocytes and melanoma cells

Diagnostic biomarkers cancer diagnostics at preclinical stage seem to be a very promising strategy to increase effectiveness of anti-cancer treatment. Currently there are no such biomarkers available for daily routine practice. However, there are some candidate molecules in research that possibly can be used as biomarkers for early diagnosis, one of them is microRNA. MicroRNA is a small, 20–25 bp, non-coding RNA that is highly involved into epigenetic regulation of gene expression. These molecules participate in malignant transformation of normal cells into cancer cells including melanoma. And moreover, definite expression level of some microRNAs are essential for normal differentiation and function of human cells. Changes in microRNA profile are one of the reasons for malignant tumor development. Identification of these changes may help to develop diagnostic systems to start anti-cancer treatment at early stages.

Application of next-generation sequencing in gastrointestinal and liver tumors

Malignant transformation of normal cells is associated with the evolution of genomic alterations. This concept has led to the development of molecular testing platforms to identify genomic alterations that can be targeted with novel therapies. Next generation sequencing (NGS) has heralded a new era in precision medicine in which tumor genes can be studied efficiently. Recent developments in NGS have allowed investigators to identify genomic predictive makers and hereditary mutations to guide treatment decision. The application of NGS in gastrointestinal cancers is being extensively studied but continues to face substantial challenges. In our review, we discuss various NGS platforms and highlight their role in identifying familial mutations and markers of response or resistance to cancer therapy. We also provide a balanced discussion of the challenges that limit the routine use of NGS in clinical practice.

The Role of DNA Methylation in Cancer.

The malignant transformation of normal cells is driven by both genetic and epigenetic changes. With the advent of next-generation sequencing and large-scale multinational consortium studies, it has become possible to profile the genomes and epigenomes of thousands of primary tumors from nearly every cancer type. From these genome-wide studies, it became clear that the dynamic regulation of DNA methylation is a critical epigenetic mechanism of cancer initiation, maintenance, and progression. Proper control of DNA methylation is not only crucial for regulating gene transcription, but its broader consequences include maintaining the integrity of the genome and modulating immune response. Here, we describe the aberrant DNA methylation changes that take place in cancer and how they contribute to the disease phenotype. Further, we highlight potential clinical implications of these changes in the context of prognostic and diagnostic biomarkers, as well as therapeutic targets.

BAD-mediated apoptotic pathway is associated with human cancer development.

The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.

Processing On-the-Go in Exosomes

Breast cancer cells secrete exosomes carrying microRNA-processing proteins that promote malignant transformation in normal cells.

Abstract 2748: Aurora A kinase inhibitor MLN8237 suppresses neurosphere proliferation of pediatric glioblastoma and prolongs animal survival in patient tumor-derived orthotopic xenograft mouse model

Abstract Background: Pediatric glioblastoma (GBM) is highly aggressive and development of novel therapy is needed. Aurora A kinase is critical for mitosis and its overexpression results in malignant transformation of normal cells. MLN8237 (Alisertib) is the first orally available reversible selective inhibitor of the Aurora A serine/threonine kinase. We investigated the effects of MLN8237 on glioma stem cell proliferation and animal survival, as well as the effects on clinically-proven therapy resistant GBM cells. Methods: Levels of Aurora A kinase expression were screened in a panel of patient tumor-derived orthotopic xenograft mouse models. Anti-tumor effects was examined by Cell Counting Kit-8 assay in paired monolayer cell lines (in FBS-based media) and neurosphere cultures (in serum-free media containing EGF and bFGF) from two xenograft mouse models, IC-4687GBM (previously untreated) and IC-R0315GBM (established from a therapy-resistant autopsy sample). Therapeutic efficacy was examined in vivo by orally administered MLN8237 (30 mg/kg/day for 12 days) in NOD/SCID mice (n=10 per group) two weeks post intra-cerebral tumor cell implantation. Tumor growth was examined by MRI and histopathological analysis. Results: Overexpression of Aurora A kinase mRNA (4-10 fold over normal control) was observed in our pediatric GBM models. Tumor growth in FBS medium was suppressed in the previously untreated line (4687GBM) but not in the therapy-resistant line (R0315GBM). Cell growth of neurospheres, which are enriched with cancer stem cells, was suppressed in both GBM lines in a time- and dose-dependent manner. In IC-4687GBM, suppression of xenograft tumor growth was confirmed by MRI and histopathological analysis along with significant prolongation of animal survival in the treatment group. In the therapy-resistant model IC-R0315GBM, no significant suppression was observed, which correlates with the in vitro lack of effect on the monolayer cells. Conclusion: We detected high levels Aurora A kinase expression in pediatric GBM models and demonstrated the efficacy of MLN8237 in targeting GBM in vitro and in vivo. Our data in the therapy-resistant model IC-R0315GBM suggests the need for combination treatment in therapy-resistant GBMs. Our results support the evaluation of MLN8237 as a novel agent for pediatric GBMs. Citation Format: Mari Kogiso, Linna Zhang, Lin Qi, Holly B. Lindsay, Frank Y. Lin, Stacey Berg, Xiao-Nan Li, Jodi A. Muscal. Aurora A kinase inhibitor MLN8237 suppresses neurosphere proliferation of pediatric glioblastoma and prolongs animal survival in patient tumor-derived orthotopic xenograft mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2748. doi:10.1158/1538-7445.AM2014-2748