microRNAs are key regulators of the human transcriptome across a number of diverse biological processes, such as development, aging and cancer, where particular miRNAs have been identified as tumour suppressive and oncogenic. In this work, we elucidate, in a comprehensive manner, across 15 epithelial cancer types comprising 7316 clinical samples from the Cancer Genome Atlas, the association of miRNA expression and target regulation with the phenotypic hallmarks of cancer. Utilising penalised regression techniques to integrate transcriptomic, methylation and mutation data, we find evidence for a complex map of interactions underlying the relationship of miRNA regulation and the hallmarks of cancer. This highlighted high redundancy for the oncomiR-1 cluster of oncogenic miRNAs, in particular hsa-miR-17-5p. In addition, we reveal extensive miRNA regulation of tumour suppressor genes such as PTEN, FAT4 and CDK12, uncovering an alternative mechanism of repression in the absence of mutation, methylation or copy number changes.

Functional disruptions of susceptibility genes by large genomic structure variant (SV) deletions in germlines are known to be associated with cancer risk. However, few studies have been conducted to systematically search for SV deletions in breast cancer susceptibility genes. We analysed deep (> 30x) whole-genome sequencing (WGS) data generated in blood samples from 128 breast cancer patients of Asian and European descent with either a strong family history of breast cancer or early cancer onset disease. To identify SV deletions in known or suspected breast cancer susceptibility genes, we used multiple SV calling tools including Genome STRiP, Delly, Manta, BreakDancer and Pindel. SV deletions were detected by at least three of these bioinformatics tools in five genes. Specifically, we identified heterozygous deletions covering a fraction of the coding regions of BRCA1 (with approximately 80kb in two patients), and TP53 genes (with ∼1.6 kb in two patients), and of intronic regions (∼1 kb) of the PALB2 (one patient), PTEN (three patients) and RAD51C genes (one patient). We confirmed the presence of these deletions using real-time quantitative PCR (qPCR). Our study identified novel SV deletions in breast cancer susceptibility genes and the identification of such SV deletions may improve clinical testing.

AIMS: To identify calcifying epithelial odontogenic tumour (CEOT) mutations in oncogenes and tumour suppressor genes.
METHODS: A panel of 50 genes commonly mutated in cancer was sequenced in CEOT by next-generation sequencing. Sanger sequencing was used to cover the region of the frameshift deletion identified in one sample.
RESULTS: Missense single nucleotide variants (SNVs) with minor allele frequency (MAF) <1% were detected in
CONCLUSION: CEOT harbours mutations in the tumour suppressor

ONTD (3-Oxo-29-noroleana-1,9(11),12-trien-2,20-dicarbonitrile) is a novel synthetic derivative of glycyrrhetinic acid (GA), which has been reported to exhibit anti-inflammatory and anti-tumor activities through its mechanisms are not fully understood. Previously, we demonstrated that ONTD induces apoptosis of human hepatoma cells via a MAPK-dependent mitochondrial pathway. Recently, ONTD was found to increase sub-G1 accumulation and Annexin-V positive staining, indicating apoptotic induction effect. It was also be found that ONTD increase the PPAR-γ activity, reduce the phosphorylation of Akt and increase phosphatase and tensin homologue (PTEN) protein expression in hepatocellular carcinoma (HCC) Bel-7402 cells, and these were associated with the inhibition of cells proliferation. More importantly, these effects could be diminished by GW9662, a specific PPAR-γ antagonist, suggesting that ONTD can act as a ligand of PPAR-γ. Taken together, our novel observations suggested that ONTD may have potential implication in HCC prevention and treatment, and showed for the first time that the anti-tumor effect of ONTD may also be mediated through modulation of the PPAR-γ activation and mediated by the PTEN/Akt signaling pathway. The present study also supports ONTD as a potential drug candidate for chemoprevention or chemotherapy of HCC.

Importance: Molecular aberrations in the phosphatidylinositol-3-kinase (PI3K) pathway drive tumorigenesis. Frequently co-occurring alterations in hormone receptors and/or human epidermal growth factor receptor 2 (HER2) may be relevant to mechanisms of response and resistance.
Objective: To identify patterns of aberration in the PI3K and interactive pathways that might lead to targeted therapy opportunities in clinical practice.
Design, Setting, and Participants: From January 2013 through December 2014, 19 784 consecutive tumor samples (>40 cancer types) were sent from thousands of clinicians in 60 countries to a single commercial laboratory for molecular profiling, including next generation sequencing, protein expression (immunohistochemical analysis [IHC]), and gene amplification (fluorescent in situ hybridization or chromogenic in situ hybridization).
Main Outcomes and Measures: Patterns in targetable genomic and proteomic alterations in the PI3K pathway and coincidence with hormone receptor and HER2 alterations.
Exposures: Molecular profiling across solid tumors.
Results: Overall, 38% of patients had an alteration in 1 or more PI3K pathway components, most commonly phosphatase and tensin homologue (PTEN) loss (by IHC) (30% of all patients), followed by mutations in PIK3CA (13%), PTEN (6%), or AKT1 (1%). Seventy percent of patients with endometrial cancer and more than 50% of patients with breast, prostate, anal, hepatocellular, colorectal, and cervical cancer exhibited alterations in at least 1 PI3K pathway gene and/or gene product. Examples of frequent aberrations included PTEN loss in hepatocellular (57% of patients), colorectal (48%), gastric (36%), prostate (52%), and endometrial cancer (49%); PIK3CA mutations in endometrial (37%), breast (31%), cervical (29%), and anal cancer (27%). PIK3CA, PTEN, and AKT1 mutations occurred more frequently in the presence of hormone receptor overexpression (androgen, progesterone, or estrogen receptor). PIK3CA mutations were also more common in the HER2-positive than in the HER2-negative group; the opposite pattern was seen for PTEN mutation or PTEN loss.
Conclusions and Relevance: PI3K pathway aberrations are among the most common in cancer. They do not segregate by classic cancer histologic characteristics. Patterns of biomarker coalterations involving HER2 and hormone receptors may be important for optimizing combination treatments across cancer types.

Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl.

The poor prognosis of esophageal squamous cell carcinoma (ESCC) is mainly attributed to higher lymphatic-related metastatic ability. Whether the loss of expression of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is associated with lymphatic-related metastasis needs elucidation. In the present study, we assessed the mRNA and protein level of PTEN in ESCC by qRT-PCR and immunohistochemistry. The results showed PTEN mRNA level in tumors was significantly lower than that in corresponding non-tumor esophageal epitheliums (p<0.001), while 38 (51.4%) tumor samples were negative for expression of PTEN in ESCC tumors. Then the association between negative expression of PTEN and lymphatic-related metastasis (lymph node metastasis/3-year postoperative lymphatic metastatic recurrence) was evaluated. The proportion of PTEN-negative expression was significantly higher in positive lymph node metastasis (pN+) than that in negative lymph node metastasis (pN0) (p=0.021). The negative expression of PTEN was not an independent risk factor for the lymphatic recurrence rate in multivariate analysis (p=0.498), however, the lymphatic recurrence rate (60.5%) in PTEN-negative expression group was higher than that (36.1%) in PTEN-positive expression group (p=0.019). Furthermore, PTEN expression was stably silenced by lentiviral-vectored shRNA (Lenti-shRNA) in Eca109 (ESCC-derived cell line) to study functional effect of PTEN in vitro and in vivo. The laboratory study indicated increased cell proliferation, migration and invasion in vitro and more rapid growth rate of xenograft tumors in vivo after stable silencing of PTEN expression. Moreover, we proved that FAK/pFAK were not the main factors mediating the mechanism of metastasis in ESCC. In conclusion, negative expression of PTEN could be a useful biomarker to predict high risk for lymphatic-related metastasis in ESCC.

Breast cancer is the leading cause of cancer deaths in females worldwide occurring in both hereditary and sporadic forms. Women with inherited pathogenic mutations in the BRCA1 or BRCA2 genes have up to an 85% risk of developing breast cancer in their lifetimes. These patients are candidates for risk-reduction measures such as intensive radiological screening, prophylactic surgery or chemoprevention. However, only about 20% of familial breast cancer cases are attributed to mutations in BRCA1 and BRCA2, while a further 5-10% are attributed to mutations in other rare susceptibility genes such as TP53, STK11, PTEN, ATM and CHEK2. A multitude of genome wide association studies (GWAS) have been conducted confirming low-risk common variants associated with breast cancer in excess of 90 loci, which may contribute to a further 23% of the heritability. We currently find ourselves in "the next generation", with technologies offering deep sequencing at a fraction of the cost. Starting off primarily in a research setting, multi-gene panel testing is now utilized in the clinic to sequence multiple predisposing genes simultaneously (otherwise known as multi-gene panel testing). In this review, we focus on the hereditary breast cancer discoveries, techniques and the challenges we face in this complex disease, especially in the light of the vast amount of data we now have at hand. It has been 20 years since the first breast cancer susceptibility gene has been discovered and there has been substantial progress in unraveling the genetic component of the disease. However, hereditary breast cancer remains a challenging topic subject to common debate.

Complex molecular changes that occur during prostate cancer (PCa) progression have been described recently. Whole genome sequencing of primary PCa samples has identified recurrent gene deletions and rearrangements in PCa. Specifically, these molecular events disrupt the gene loci of phosphatase and tensin homolog (PTEN) and membrane-associated guanylate kinase inverted-2 (MAGI2). In the present study, we analyzed the expression profile of MAGI2 gene in a cohort of clinical PCa (n = 45) and benign prostatic hyperplasia (BPH) samples (n = 36) as well as three PCa cell lines. We also studied the expression of PCa-related genes, including PTEN, NKX3.1, SPINK1, DD3, AMACR, ERG, and TMPRSS2-ERG fusion in the same samples. The expression of MAGI2 mRNA was significantly down-regulated in PC3, LNCaP and DU-145 PCa cell lines (p = 0.000), and also in clinical tumor samples (Relative expression = 0.307, p = 0.002, [95 % CI 0.002-12.08]). The expression of PTEN, NKX3.1, SPINK1, DD3, and AMACR genes was significantly deregulated in prostate tumor samples (p range 0.000-0.044). A significant correlation was observed between MAGI2 and NKX3.1 expression in tumor samples (p = 0.006). Furthermore, the inclusion of MAGI2 in the gene panel improved the accuracy for discrimination between PCa and BPH samples with the sensitivity and specificity of 0.88 [CI 0.76-0.95] and 0.83 [CI 0.68-0.92], respectively. The data presented here suggest that MAGI2 gene can be considered as a novel component of gene signatures for the detection of PCa.

MYC is one of the most frequently mutated and overexpressed genes in human cancer but the regulation of MYC expression and the ability of MYC protein to repress cellular genes (including itself) have remained mysterious. Recent genome-wide association studies show that many genetic polymorphisms associated with disease risk map to distal regulatory elements that regulate the MYC promoter through large chromatin loops. Cancer risk-associated single-nucleotide polymorphisms (SNPs) contain more potent enhancer activity, promoting higher MYC levels and a greater risk of disease. The MYC promoter is also subject to complex regulatory circuits and limits its own expression by a feedback loop. A model for MYC autoregulation is discussed which involves a signaling pathway between the PTEN (phosphatase and tensin homolog) tumor suppressor and repressive histone modifications laid down by the EZH2 methyltransferase.

The expression of miR-200c has been widely reported to be elevated in tumor tissues and sera of patients with colorectal cancer (CRC) and has been found to correlate with poor prognosis. However, how miR-200c regulates the apoptosis, survival, invasion, metastasis, and tumor growth in colon cancer cells remains to be fully elucidated. This study seeks to further investigate the role of miR-200c in colon cancer development. The expression of miR-200c in tumor and peritumoral tissues of 101 colon cancer patients was measured by real-time PCR. miR-200c expression in HCT-116 and HT-29 colon cancer cells was silenced by adenovirus-carried expression of antisense mRNA against miR-200c. The protein levels of PTEN, p53 Ser(15), PP1, and activated caspase-3 in HCT-116 and HT-29 cells were measured by Western blot. This study demonstrated that the expression of miR-200c was significantly higher in tumor tissues than in peritumoral tissues of colon cancer patients. The elevated miR-200c expression significantly correlated with the TNM stage, lymph node metastasis, and invasion of colon cancer. Silencing miR-200c expression significantly induced cell apoptosis, inhibited long-term survival, invasion, and metastasis, and delayed xenograft tumor growth. Importantly, silencing miR-200c expression sensitized the therapeutic effect of Ara-C (Cytarabine). The effects of silencing miR-200c expression were associated with upregulation of PTEN protein and p53 Ser(15) phosphorylation levels in HCT-116 cells and PTEN protein expression in HT-29 cells. In conclusion, miR-200c functions as an oncogene in colon cancer cells through regulating tumor cell apoptosis, survival, invasion, and metastasis as well as xenograft tumor growth through inhibition of PTEN expression and p53 phosphorylation.

Epithelial-mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non-small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA)-related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in NSCLC. miRNA expression profiles were examined before and after transforming growth factor β1 (TGF-β1) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. miRNA array and real-time quantitative reverse transcription PCR (qRT-PCR) revealed that TGF-β1 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW, and PDZ domain-containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN, was diminished in A549 cells with EMT after the TGF-β1 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-β1-induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-β1-induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, in which suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be a new therapeutic target in patients with advanced lung adenocarcinoma, depending on the EMT phenomenon.

The malignant mesothelioma is an aggressive form of cancer with a mean survival rate of less than a year. Moreover, environmental exposure to minerals is an important factor in the development of malignant mesothelioma (MM), especially the mineral asbestos, which has a well-documented role in MM, and more recently, the mineral erionite has been proven to be a strong carcinogenic inducer of MM. In addition, the virus simian virus 40 has been implicated as a co-carcinogenic player in MM. However, the molecular mechanisms involved in the pathogenesis of this cancer are still not fully understood. Indeed, it is known that several genes are altered or mutated in MM, among those are p16(INK4A), p14(ARF), and neurofibromatosis type II. Furthermore, TP53 has been reported to be mutated in the majority of the cancers; however, in MM, it is very uncommon mutations in this gene. Also, the PTEN gene has been shown to play an important role in endometrial cancer and glioblastoma, although the role of PTEN in MM has yet to be established. Taken altogether, this review focuses on the historical aspects, molecular mechanisms, interaction with other genes and proteins, and the role of these genes in MM. Lastly, this review questions the cancer theory of the two hits because the functions of both PTEN and TP53 are not fully explained by this theory.

The G protein-coupled estrogen receptor (GPER, GPR30) is suggested to be involved in non-nuclear estrogen signaling and is expressed in a variety of hormone dependent cancer entities. This study was performed to further elucidate the role of this receptor in endometrial adenocarcinoma. We first analyzed GPER expression at the mRNA level in 88 endometrial cancer or normal endometrial tissue samples and compared it to those of nuclear steroid hormone receptors. GPER transcript levels were found to be about 6-fold reduced, but still present in endometrial cancer. Expression of this receptor was decreased in all grading subgroups when compared to pre- or postmenopausal endometrium. GPER mRNA expression was associated with PR mRNA levels (Spearman's rho 0.4610, p<0.001). We then tested the effect of the GPER ligand G-1 on growth of three endometrial cancer cell lines with different GPER expression. GPER protein levels were highest in RL95-2 cells, moderate in HEC-1A cells and not detectable in HEC-1B cells. The moderate expression level in HEC-1A cells was similar to average tumor tissue expression. Treatment with G-1 significantly inhibited growth of the GPER-positive cell lines RL95-2 and HEC-1A in a dose-dependent manner, whereas the GPER-negative line HEC-1B was not affected. Though GPER transcript levels were found to be reduced in endometrial cancer, our in vitro data suggest that moderate GPER expression might be sufficient to mediate growth-inhibitory effects triggered by its agonist G-1.

PURPOSE: PIK3CA and PTEN mutations are prevalent in colorectal cancer and potential markers of response to mitogen-activated protein/extracellular signal-regulated kinase inhibitors and anti-EGF receptor antibody therapy. Relationships between phosphoinositide 3-kinase (PI3K) pathway mutation, clinicopathologic characteristics, molecular features, and prognosis remain controversial.
EXPERIMENTAL DESIGN: A total of 1,093 stage I-IV colorectal cancers were screened for PIK3CA (exons 9 and 20), KRAS (codons 12-13), BRAF (codon 600) mutations, and microsatellite instability (MSI). PTEN (exons 3-8) and CpG island methylator phenotype (CIMP) status were determined in 744 and 489 cases. PIK3CA data were integrated with 17 previous reports (n = 5,594).
RESULTS: PIK3CA and PTEN mutations were identified in 11.9% and 5.8% of colorectal cancers. PTEN mutation was associated with proximal tumors, mucinous histology, MSI-high (MSI-H), CIMP-high (CIMP-H), and BRAF mutation (P < 0.02). PIK3CA mutation was related to older age, proximal tumors, mucinous histology, and KRAS mutation (P < 0.04). In integrated cohort analysis, PIK3CA exon 9 and 20 mutations were overrepresented in proximal, CIMP-low (CIMP-L), and KRAS-mutated cancers (P ≤ 0.011). Comparing PIK3CA exonic mutants, exon 20 mutation was associated with MSI-H, CIMP-H, and BRAF mutation, and exon 9 mutation was associated with KRAS mutation (P ≤ 0.027). Disease-free survival for stage II/III colorectal cancers did not differ by PI3K pathway status.
CONCLUSION: PI3K pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile-serrated pathway (MSI-H/CIMP-H/BRAF(mut)), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the traditional serrated pathway (CIMP-L/KRAS(mut)) of tumorigenesis. Our data highlight the PI3K pathway as a therapeutic target in distinct colorectal cancer subtypes.

Cancer/testis (CT) antigens encoded by CT genes are immunogenic antigens, and the expression of CT gene is strictly restricted to only the testis among mature organs. Therefore, CT antigens are promising candidates for cancer immunotherapy. In a previous study, we identified a novel CT antigen, DNAJB8. DNAJB8 was found to be preferentially expressed in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), and it is thus a novel CSC antigen. In this study, we hypothesized that CT genes are preferentially expressed in CSCs/CICs rather than in non-CSCs/-CICs and we examined the expression of CT genes in CSCs/CICs. The expression of 74 CT genes was evaluated in side population (SP) cells (=CSC) and main population (MP) cells (=non-CSC) derived from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells and MCF7 breast adenocarcinoma cells by RT-PCR and real-time PCR. Eighteen genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) showed higher expression levels in SP cells than in MP cells, whereas 10 genes (BAGE1, BAGE2, BAGE4, BAGE5, XAGE1, LIP1, D40, HCA661, TDRD1 and TPTE) showed similar expression levels in SP cells and MP cells. Thus, considerable numbers of CT genes showed preferential expression in CSCs/CICs. We therefore propose a novel sub-category of CT genes in this report: cancer/testis/stem (CTS) genes.

PTEN (Phosphatase and tensin homolog) is a tumour suppressor gene commonly defective in human cancer, and is thus a potentially important therapeutic target. Targeting tumour suppressor loss-of-function is possible by exploiting the genetic concept of synthetic lethality (SL). By combining the use of isogenic models of PTEN deficiency with high-throughput RNA interference (RNAi) screening, we have identified Nemo-Like Kinase (NLK) inhibition as being synthetically lethal with PTEN deficiency. This SL is likely mediated by the transcription factor FOXO1 (Forkhead box O1), an NLK substrate, as the selectivity of NLK gene silencing for PTEN deficient cells can be reversed by FOXO1 knockdown. In addition, we provide evidence that PTEN defective cells targeted by NLK gene depletion undergo senescence, suggesting that NLK function is critical for the continued proliferation of PTEN deficient cells. Taken together, these data provide new insight into the potential of targeting of NLK to treat a range of tumourigenic conditions characterised by PTEN deficiency.

One of pathological hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs) consisting of abnormally hyperphosphorylated tau. The molecular mechanisms underlying the regulation of tau hyperphosphorylation remain largely unclear. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated in the pathogenesis of AD, however, potential functions and role of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in AD pathogenesis have not been fully explored. Here, we report that okadaic acid (OA)-induced tau phosphorylation is accompanied by PTEN induction, knockdown of PTEN reduces the tau hyperphosphorylation by OA in SH-SY5Y cells and increases cell proliferation and survival. The effect of PTEN suppression on tau dephosphorylation appeared to be mediated by inhibition of glycogen synthase kinase 3 while enhancing the Akt activity. Reduction of tau phosphorylation was also observed in the OA-induced parental SH-SY5Y cells co-treated with bisperoxovanadate (bpv), a potent PTEN inhibitor. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis and give some insight into the mechanisms through which suppression of PTEN expression may contribute towards the amelioration of tauopathy, implying that pharmacological intervention of PTEN may be a new therapeutic approach for the treatment of AD.

The identification of somatic driver mutations in human samples has allowed for the development of a molecular classification for melanoma. Recent breakthroughs in the treatment of metastatic melanoma have arisen as a result of these significant new insights into the molecular biology of the disease, particularly the development of inhibitors of activating BRAF(V600E) mutations. In this article the roles of several mutations known to be involved in the malignant transformation of melanocytes are reviewed including BRAF, PTEN, NRAS, ckit, and p16 as well as some of the emerging mutations in cutaneous and uveal melanoma. The bench to bedside collaborations that resulted in these discoveries are summarized, and potential therapeutic strategies to target driver mutations in specific patient subsets are discussed.

Cowden syndrome is a rare, autosomal-dominant, multisystem disorder characterized by hamartomatous tissue overgrowth and an increased risk of breast, thyroid, and endometrial cancers. Most of the cases arise from germline mutations of the phosphatase and tensin homologue tumor suppressor gene. An association with colon cancer remains unproven but has been suggested in previous reports. We present the case of a 42-year-old man with colonic ganglioneuromatous polyps and an adjacent colonic adenoma giving rise to a signet-ring adenocarcinoma with lymph node metastases in the setting of Cowden syndrome. Although gastrointestinal polyps are a common feature of Cowden syndrome, reports of ganglioneuromatous polyps and malignant degeneration are rare. Cutaneous features of Cowden syndrome in our patient include "cobblestone" lesions of the tongue and oral mucosa, facial trichilemmomas, multiple acral keratoses, and a storiform collagenoma.

Epstein-Barr virus (EBV) has been accepted as an infective agent causing gastric carcinoma (GC). EBV-associated GC, comprising nearly 10% of all cases of GC, is the monoclonal growth of EBV-infected epithelial cells, which express only several EBV-latent genes (Latency I program). Histopathologically, there are two subtypes, lymphoepithelioma-like carcinoma and the ordinary type of GC. Other features include the lace pattern of carcinoma cells in the intramucosal stage and the dense infiltration of lymphocytes and macrophages at the invasive site of the submucosa. The primary molecular abnormality in EBV-associated GC is global and non-random CpG island methylation in the promoter region of many cancer-related genes. Experimental studies have demonstrated that viral latent membrane protein 2A (LMP2A) is responsible for the promotion of DNA methylation. LMP2A up-regulates cellular DNMT1 through the phosphorylation of STAT3, resulting in the repression of tumor suppressor genes, such as PTEN, through promoter methylation. DNA methylation in EBV-infected stomach cells may be due to overdrive of the cellular defense against foreign DNA. Further studies on the mechanisms of epigenetic abnormalities will clarify the strategies for prevention and treatment of this particular type of GC with EBV infection.

WNT, RAS or phosphoinositide 3-kinase signaling pathways control specific stages of ovarian follicular development. To analyze the functional interactions of these pathways in granulosa cells during follicular development in vivo, we generated specific mutant mouse models. Stable activation of the WNT signaling effector β-catenin (CTNNB1) in granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life or following targeted deletion of the tumor suppressor gene Pten. Conversely, expression of oncogenic KRAS(G12D) dramatically arrests proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRAS(G12D) in granulosa cells, we sought to determine whether KRAS(G12D) would block precancerous lesion and tumor formation in follicles of the CTNNB1-mutant mice. Unexpectedly, transgenic Ctnnb1;Kras-mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation and developed early-onset GCTs leading to premature death in a manner similar to the Ctnnb1;Pten-mutant mice. Microarray and reverse transcription-PCR analyses revealed that gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. The concomitant activation of CTNNB1 and KRAS in Sertoli cells also caused testicular granulosa cell tumors that showed gene expression patterns that partially overlapped those observed in GCTs of the ovary. Although the mutations analyzed herein have not yet been linked to adult GCTs in humans, they may be related to juvenile GCTs or to tumors in other tissues where CTNNB1 is mutated. Importantly, the results provide strong evidence that CTNNB1 is the driver in these contexts and that KRAS(G12D) and Pten loss promote the program set in motion by the CTNNB1.

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

An important molecular target for metastatic CRC treatment is the epidermal growth factor receptor (EGFR). Many potential biomarkers predictive of response to anti-EGFR monoclonal antibodies (cetuximab and panitumumab) have been retrospectively evaluated, including EGFR activation markers and EGFR ligands activation markers. With regard to the "negative predictive factors" responsible for primary or intrinsic resistance to anti-EGFR antibodies a lot of data are now available. Among these, KRAS mutations have emerged as a major predictor of resistance to panitumumab or cetuximab in the clinical setting and several studies of patients receiving first and subsequent lines of treatment have shown that those with tumors carrying KRAS mutations do not respond to EGFR-targeted monoclonal antibodies or show any survival benefit from such treatments. The role of B-RAF mutations, mutually exclusive with KRAS mutations, in predicting resistance to anti-EGFR mAbs is not yet consolidated. It therefore appears that BRAF mutations may play a strong negative prognostic role and only a slight role in resistance to anti-EGFR Abs.

The transcription factor EGR1 is a tumor suppressor gene that is downregulated in many cancer types. Clinically, loss of EGR1 translates to increased tumor transformation and subsequent patient morbidity and mortality. In synovial sarcoma, the SS18-SSX fusion protein represses EGR1 expression through a direct association with the EGR1 promoter. However, the mechanism through which EGR1 becomes downregulated in other tumor types is unclear. Here, we report that EGR1 is regulated by microRNA (miR)-183 in multiple tumor types including synovial sarcoma, rhabdomyosarcoma (RMS), and colon cancer. Using an integrative network analysis, we identified that miR-183 is significantly overexpressed in these tumor types as well as in corresponding tumor cell lines. Bioinformatic analyses suggested that miR-183 could target EGR1 mRNA and this specific interaction was validated in vitro. miR-183 knockdown in synovial sarcoma, RMS, and colon cancer cell lines revealed deregulation of a miRNA network composed of miR-183-EGR1-PTEN in these tumors. Integrated miRNA- and mRNA-based genomic analyses indicated that miR-183 is an important contributor to cell migration in these tumor types and this result was functionally validated to be occurring via an EGR1-based mechanism. In conclusion, our findings have significant implications in the mechanisms underlying EGR1 regulation in cancers. miR-183 has a potential oncogenic role through the regulation of 2 tumor suppressor genes, EGR1 and PTEN, and the deregulation of this fundamental miRNA regulatory network may be central to many tumor types.

The psiTPTE22 gene has been designated as a TPTE pseudogene. Our study found that the 5' part of psiTPTE22 has no sequence similarity to TPTE and contains a 3.8-kb human endogenous retrovirus (HERV) element. Because of the HERV element, the 5' part of psiTPTE22 (psiTPTE22-HERV) expresses independently as a gene. Comparison between the DNA sequences of humans and chimps indicated that psiTPTE22-HERV is human specific. We identified 3 alternatively spliced transcript variants from psiTPTE22-HERV by a PCR-based strategy, which use the transcriptional termination signal contained in the HERV element. A 402-nt ORF was contained in the 2 longer transcripts. Western blotting using antibodies produced with chemically synthesized peptide confirmed that a 15-kDa protein was translated from this ORF. RT-PCR results indicated that the ORF-containing transcripts were mainly expressed in psiTPTE22-HERV-expressing samples. Real-time quantitative RT-PCR results showed that expression of the 402-nt ORF was upregulated in normal tissues of kidney, liver, stomach, and lung but downregulated in corresponding tumor tissues. This gene is located near the centromere of chromosome 22 and has a high GC content around the promoter region. Bisulfite sequencing PCR results indicated that it is silenced in cancers by DNA methylation. The expression of psiTPTE22-HERV can be recovered in cancer cells using DNA methylation and histone deacetylase inhibitors. These results suggest psiTPTE22-HERV is regulated epigenetically by DNA methylation. Our study paved the way for further study on an interesting HERV-related human-specific gene, which is silenced in cancers by DNA methylation.

Malignant gliomas are the most common primary brain tumors. Despite efforts to find effective treatments, these tumors remain incurable. The failure of malignant gliomas to respond to conventional cancer therapies may reflect the unique biology of these tumors, underscoring the need for new approaches in their investigation. Recently, progress has been made in characterization of the molecular pathogenesis of glioblastoma using a developmental neurobiological perspective, by exploring the role of signaling pathways that control the differentiation of neural stem cells along the glial lineage. The transcription factor STAT3, which has an established function in neural stem cell and astrocyte development, has been found to play dual tumor suppressive and oncogenic roles in glial malignancy depending on the mutational profile of the tumor. These findings establish a novel developmental paradigm in the study of glioblastoma pathogenesis and provide the rationale for patient-tailored therapy in the treatment of this devastating disease.

Micro RNAs (miRNAs) are non-coding small RNAs and constitute a novel class of negative gene regulators that are found in both plants and animals. Several miRNAs play crucial roles in cancer cell growth. To identify miRNAs specifically deregulated in anaplastic thyroid cancer (ATC) cells, we performed a comprehensive analysis of miRNA expressions in ARO cells and primary thyrocytes using miRNA microarrays. MiRNAs in a miR-17-92 cluster were overexpressed in ARO cells. We confirmed the overexpression of those miRNAs by Northern blot analysis in ARO and FRO cells. In 3 of 6 clinical ATC samples, miR-17-3p and miR-17-5p were robustly overexpressed in cancer lesions compared to adjacent normal tissue. To investigate the functional role of these miRNAs in ATC cells, ARO and FRO cells were transfected with miRNA inhibitors, antisense oligonucleotides containing locked nucleic acids. Suppression of miR-17-3p caused complete growth arrest, presumably due to caspase activation resulting in apoptosis. MiR-17-5p or miR-19a inhibitor also induced strong growth reduction, but only miR-17-5p inhibitor led to cellular senescence. On the other hand, miR-18a inhibitor only moderately attenuated the cell growth. Thus, we have clarified functional differences among the members of the cluster in ATC cells. In conclusion, these findings suggest that the miR-17-92 cluster plays an important role in certain types of ATCs and could be a novel target for ATC treatment.

Medulloblastoma is the most common childhood malignant tumor of the central nervous system. Treatment of medulloblastoma requires harmful therapy and nevertheless carries a poor prognosis. Due to their presence in various cancers and their limited expression in normal tissues, CT antigens are ideal vaccine targets for tumor immunotherapy. CT antigens, such as MAGE and NY-ESO-1, have been employed in clinical trials in various malignancies but little is known about their presence in medulloblastoma. We analyzed 25 medulloblastomas for the expression of a panel of CT antigens by RT-PCR and immunohistochemistry. Messenger RNA expression in the samples was as follows: GAGE 64%, MAGEA3/6 56%, SYCP1 44%, SLCO6A1 32%, MAGEC1 28%, MAGEC2 28%, MAGEA4 28%, NY-ESO-1 20%, MAGEA1 16%, and TPTE 0%. All cases except one (96%) were positive for mRNA expression of at least one CT gene. However, CT antigen expression was scarce on a protein level. Immunoreaction to monoclonal antibody E978 (NY-ESO-1) was negative in all cases; MA454 (MAGEA1), 57B (MAGEA4), M3H67 (MAGEA3/6), CT10#5 (MAGEC2) and #23 (GAGE) were each positive in 1 case, while the highest incidence of positive immunostaining, albeit heterogeneous, was seen with CT7-33 (MAGEC1) in 3 out of the 25 cases. The absence of correlation between mRNA and protein expression in medulloblastoma has not been observed in other tumors and further studies addressing the biology of CT antigens are necessary to investigate the present discrepant results.

Previously, we have developed a unique in vitro LNCaP cell model, which includes androgen-dependent (LNCaP-C33), androgen-independent (LNCaP-C81) and an intermediate phenotype (LNCaP-C51) cell lines resembling the stages of prostate cancer progression to hormone independence. This model is advantageous in overcoming the heterogeneity associated with the prostate cancer up to a certain extent. We characterized and compared the gene expression profiles in LNCaP-C33 (androgen-dependent) and LNCaP-C81 (androgen-independent) cells using Affymetrix GeneChip array analyses. Multiple genes were identified exhibiting differential expression during androgen-independent progression. Among the important genes upregulated in androgen-independent cells were PCDH7, TPTE, TSPY, EPHA3, HGF, MET, EGF, TEM8, etc., whereas many candidate tumor suppressor genes (HTATIP2, CDKN2A, CDKN2B, CDKN1C, TP53, TP73, ICAM1, SOCS1/2, SPRY2, PPP2CA, PPP3CA, etc.) were decreased. Pathway prediction analysis identified important gene networks associated with growth-promoting and apoptotic signaling that were perturbed during androgen-independent progression. Further investigation of one of the genes, PPP2CA, which encodes the catalytic subunit of a serine phosphatase PP2A, a potent tumor suppressor, revealed that its expression was decreased in prostate cancer compared to adjacent normal/benign tissue. Furthermore, the downregulated expression of PPP2CA was significantly correlated with tumor stage and Gleason grade. Future studies on the identified differentially expressed genes and signaling pathways may be helpful in understanding the biology of prostate cancer progression and prove useful in developing novel prognostic biomarkers and therapy for androgen-refractory prostate cancer.