Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required.

Unlike epigenetic silencing of tumor suppressor genes, the role of epigenetic derepression of cancer-promoting genes or oncogenes in carcinogenesis remains less well understood. The tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer and their overexpression was previously reported to promote the migration and invasion of ovarian epithelial cells. Here, we show that the expression of claudin-3 and claudin-4 is repressed in ovarian epithelial cells in association with promoter 'bivalent' histone modifications, containing both the activating trimethylated histone H3 lysine 4 (H3K4me3) mark and the repressive mark of trimethylated histone H3 lysine 27 (H3K27me3). During ovarian tumorigenesis, derepression of CLDN3 and CLDN4 expression correlates with loss of H3K27me3 in addition to trimethylated histone H4 lysine 20 (H4K20me3), another repressive histone modification. Although CLDN4 repression was accompanied by both DNA hypermethylation and repressive histone modifications, DNA methylation was not required for CLDN3 repression in immortalized ovarian epithelial cells. Moreover, activation of both CLDN3 and CLDN4 in ovarian cancer cells was associated with simultaneous changes in multiple histone modifications, whereas H3K27me3 loss alone was insufficient for their derepression. CLDN4 repression was robustly reversed by combined treatment targeting both DNA demethylation and histone acetylation. Our study strongly suggests that in addition to the well-known chromatin-associated silencing of tumor suppressor genes, epigenetic derepression by the conversely related loss of repressive chromatin modifications also contributes to ovarian tumorigenesis via activation of cancer-promoting genes or candidate oncogenes.

Therapies targeting SRC family kinases (SFKs) have shown efficacy in treating non-small cell lung cancer (NSCLC). However, recent clinical trials have found that the SFK inhibitor dasatinib is ineffective in some patient cohorts. Regardless, dasatinib treatment may benefit some NSCLC patient subgroups. Here, we investigated whether expression of LYN, a member of the SFK family, is associated with patient survival, the efficacy of dasatinib, and/or NSCLC cell viability. LYN expression was associated with poor overall survival in a multivariate analysis, and this association was strongest in non-smoker female patients with adenocarcinoma (ADC). In lung ADC cells, LYN expression enhanced cell proliferation, migration, and invasion. Dasatinib inhibited LYN activity and decreased cell viability in LYN-positive ADC cell lines and xenografts. Additionally, we identified the SFKs SRC and YES as candidate dasatinib targets in LYN-negative ADC cell lines. Our findings suggest that LYN is a useful prognostic marker and a selective target of dasatinib therapy in the lung ADC subpopulation especially in female non-smokers with lung ADC.

Rhabdomyosarcoma (RMS) is the most commonly occurring type of soft tissue tumor in children. However, it is rare in adults, and therefore, very little is known about the most appropriate treatment strategy for adult RMS patients. We performed genomic analysis of RMS cells derived from a 27-year-old male patient whose disease was refractory to treatment. A peritoneal seeding nodule from the primary tumor, pleural metastases, malignant pleural effusion, and ascites obtained during disease progression, were analyzed. Whole exome sequencing revealed 23 candidate variants, and 10 of 23 mutations were validated by Sanger sequencing. Three of 10 mutations were present in both primary and metastatic tumors, and 3 mutations were detected only in metastatic specimens. Comparative genomic hybridization array analysis revealed prominent amplification in the 12q13-14 region, and more specifically, the CDK4 proto-oncogene was highly amplified. ALK overexpression was observed at both protein and RNA levels. However, an ALK fusion assay using NanoString technology failed to show any ALK rearrangements. Little genetic heterogeneity was observed between primary and metastatic RMS cells. We propose that CDK4, located at 12q14, is a potential target for drug development for RMS treatment.

Malignant pheochromocytoma/paraganglioma (PCC/PGL) is defined by the presence of metastases at non-chromaffin sites, which makes it difficult to prospectively diagnose malignancy. Here, we performed array CGH (aCGH) and paired gene expression profiling of fresh, frozen PCC/PGL samples (n = 12), including three malignant tumors, to identify genes that distinguish benign from malignant tumors. Most PCC/PGL cases showed few copy number aberrations, regardless of malignancy status, but mRNA analysis revealed that 390 genes were differentially expressed in benign and malignant tumors. Expression of the enzyme, phenylethanolamine N-methyltransferase (PNMT), which catalyzes the methylation of norepinephrine to epinephrine, was significantly lower in malignant PCC/PGL as compared to benign samples. In 62 additional samples, we confirmed that PNMT mRNA and protein levels were decreased in malignant PCC/PGL using quantitative real-time polymerase chain reaction and immunohistochemistry. The present study demonstrates that PNMT downregulation correlates with malignancy in PCC/PGL and identifies PNMT as one of the most differentially expressed genes between malignant and benign tumors.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by poor patient prognosis and for which no targeted therapies are currently available. TNBC can be further categorized as either basal-like (BLBC) or quintuple-negative breast cancer (QNBC). In the present study, we aimed to identify novel molecular therapeutic targets for TNBC by analyzing the mRNA expression of TNBC-related genes in publicly available microarray data sets. We found that Engrailed 1 (EN1) was significantly overexpressed in TNBC. Using breast cancer cell lines, we found that EN1 was more highly expressed in TNBC than in other breast cancer subtypes. EN1 expression was analyzed in 199 TNBC paraffin-embedded tissue samples by immunohistochemistry. EN1 protein expression was positively associated with reduced overall survival (OS) rate in patients with QNBC, but not those with BLBC. The importance of EN1 expression in QNBC cell viability and tumorigenicity was evaluated using the QNBC cell lines, HCC38 and HCC1395. Based on our data, EN1 may promote the proliferation, migration, and multinucleation of QNBC cells, likely via the transcriptional activation of HDAC8, UTP11L, and ZIC3. We also demonstrated that actinomycin D effectively inhibits EN1 activity in QNBC cells. The results of the present study suggest that EN1 activity is highly clinically relevant to the survival prognosis of patients with QNBC and EN1 is a promising potential therapeutic target for future QNBC treatment.

Triple-negative breast cancer is characterized by the absence of estrogen and progesterone receptors and human epidermal growth factor receptor 2, and is associated with a poorer outcome than other subtypes of breast cancer. Moreover, there are no accurate prognostic genes or effective therapeutic targets, thereby necessitating continued intensive investigation. This study analyzed the genetic mutation landscape in 70 patients with triple-negative breast cancer by targeted exome sequencing of tumor and matched normal samples. Sequencing showed that more than 50% of these patients had deleterious mutations and homozygous deletions of DNA repair genes, such as ATM, BRCA1, BRCA2, WRN, and CHEK2. These findings suggested that a large number of patients with triple-negative breast cancer have impaired DNA repair function and that therefore a poly ADP-ribose polymerase inhibitor may be an effective drug in the treatment of this disease. Notably, homozygous deletion of three genes, EPHA5, MITF, and ACSL3, was significantly associated with an increased risk of recurrence or distant metastasis in adjuvant chemotherapy-treated patients.

The purpose of this study was to identify genotypes associated with dose-adjusted tacrolimus trough concentrations (C0/D) in kidney transplant recipients using whole-exome sequencing (WES). This study included 147 patients administered tacrolimus, including seventy-five patients in the discovery set and seventy-two patients in the replication set. The patient genomes in the discovery set were sequenced using WES. Also, known tacrolimus pharmacokinetics-related intron variants were genotyped. Tacrolimus C0/D was log-transformed. Sixteen variants were identified including novel CYP3A7 rs12360 and rs10211 by ANOVA. CYP3A7 rs2257401 was found to be the most significant variant among the periods by ANOVA. Seven variants including CYP3A7 rs2257401, rs12360, and rs10211 were analyzed by SNaPshot in the replication set and the effects on tacrolimus C0/D were verified. A linear mixed model (LMM) was further performed to account for the effects of the variants and clinical factors. The combined set LMM showed that only CYP3A7 rs2257401 was associated with tacrolimus C0/D after adjusting for patient age, albumin, and creatinine. The CYP3A7 rs2257401 genotype variant showed a significant difference on the tacrolimus C0/D in those expressing CYP3A5, showing its own effect. The results suggest that CYP3A7 rs2257401 may serve as a significant genetic marker for tacrolimus pharmacokinetics in kidney transplantation.

Chronic exposure to TGFβ, a frequent occurrence for tumor cells in the tumor microenvironment, confers more aggressive phenotypes on cancer cells by promoting their invasion and migration while at the same time increasing their resistance to the growth-inhibitory effect of TGFβ. In this study, a transdifferentiated (TD) A549 cell model, established by chronically exposing A549 cells to TGFβ, showed highly invasive phenotypes in conjunction with attenuation of Smad-dependent signaling. We show that Snail protein, the mRNA expression of which strongly correlates with a poor prognosis in lung cancer patients, was highly stable in TD cells after TGFβ stimulation. The increased protein stability of Snail in TD cells correlated with elevated inhibitory phosphorylation of GSK3β, resulting from the high Akt activity. Notably, integrin β3, whose expression was markedly increased upon sustained exposure to TGFβ, was responsible for the high Akt activity as well as the increased Snail protein stability in TD cells. Consistently, clinical database analysis on lung cancer patients revealed a negative correlation between overall survival and integrin β3 mRNA levels. Therefore, we suggest that the integrin β3-Akt-GSK3β signaling axis plays an important role in non-canonical TGFβ signaling, determining the invasive properties of tumor cells chronically exposed to TGFβ.

The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

Toxicity and resistance remain major challenges for advanced or recurrent cervical cancer therapies, as treatment requires high doses of chemotherapeutic agents. Restoration of TP53 and hypophosphorylated-retinoblastoma (pRB) proteins by human papillomavirus (HPV) E6/E7 siRNA sensitizes HPV-positive cervical cancer cells toward chemotherapeutic agents. Here, we investigated the therapeutic effects of E6/E7 siRNA on the dynamic behavior of TP53 and RB/E2F signaling networks in deciding the cell fate. The synergistic effect of HPV E6/E7 siRNA pool (SP) with chemotherapeutic agents on TP53 and RB/E2F signaling, proliferation, and apoptosis was analyzed in vitro and in vivo. Compared to the E6/E7 SP alone, E6/E7 SP with cisplatin treatment effectively restored TP53 and RB/E2F signaling and contributes to differences in cell fate, such as apoptosis or cell cycle arrest. We also developed a cellular dynamics model that includes TP53-RB/E2F dynamics and cell proliferation profiles, and confirmed its utility for investigating E6/E7 siRNA-based combination regimens. Using a dual reporter system, we further confirmed the cross talk between TP53 and RB/E2F signaling mechanisms. Treatment of E6/E7 SP cationic liposome (i.v.) with cisplatin and paclitaxel (i.p.) potentially inhibited tumor growth in BALB/c-nude mice. Altogether, our findings suggest that stabilization of TP53 and the RB/E2F repressor complex by E6/E7 SP combined with low-dose chemotherapy can effectively suppress tumor growth.

Despite significant improvements in colon cancer outcomes over the past few decades, preclinical development of more effective therapeutic strategies is still limited by the availability of clinically relevant animal models. To meet those clinical unmet needs, we generated a well-characterized in vivo preclinical platform for colorectal cancer using fresh surgical samples.

Surgery and radiation are the current standard treatments for cervical cancer. However, there is no effective therapy for metastatic or recurrent cases, necessitating the identification of therapeutic targets. In order to create preclinical models for screening potential therapeutic targets, we established 14 patient-derived xenograft (PDX) models of cervical cancers using subrenal implantation methods. Serially passaged PDX tumors retained the histopathologic and genomic features of the original tumors. Among the 9 molecularly profiled cervical cancer patient samples, a HER2-amplified tumor was detected by array comparative genomic hybridization and targeted next-generation sequencing. We confirmed HER2 overexpression in the tumor and serially passaged PDX. Co-administration of trastuzumab and lapatinib in the HER2-overexpressed PDX significantly inhibited tumor growth compared to the control. Thus, we established histopathologically and genomically homologous PDX models of cervical cancer using subrenal implantation. Furthermore, we propose HER2 inhibitor-based therapy for HER2-amplified cervical cancer refractory to conventional therapy.

Solitary fibrous tumors (SFTs) are NAB2-STAT6 fusion-associated neoplasms. There are several subtypes of NAB2-STAT6 fusions, but their clinical significances are still unclear. Moreover, the mechanisms of malignant progression are also poorly understood. In this study, using 91 SFT cases, we examined whether fusion variants are associated with clinicopathological parameters and also investigated the molecular mechanism of malignant transformation using whole-exome sequencing. We detected variant 1b (NAB2ex4-STAT6ex2) in 51/91 (56%) cases and variants 2a/2b (NAB2ex6-STAT6ex16/17) in 17/91 (19%) cases. The NAB2-STAT6 fusion variant types were significantly associated with their primary site (P < 0.001). In addition, a TERT promoter mutation was detected in 7/73 (10%) cases, and it showed a significant association with malignant SFTs (P = 0.003). To identify molecular changes during malignant progression, we selected an index patient to obtain parallel tissue samples from the primary and metastatic tumors. In the metastatic tissue, 10 unique molecular alterations, including those in TP53 and APAF1, were detected. In vitro functional experiments showed that APAF1 depletion increased the tumor potency of cells expressing NAB2-STAT6 fusion protein under treatment with staurosporine. We found that TP53 immunopositivity (P = 0.006) and loss of APAF1 immunoreactivity (P < 0.001) were significantly associated with malignant SFTs. Our study suggests that dysfunction of TP53 and APAF1 leads to impaired apoptotic function, and eventually contributes toward malignant SFT transformation. KEY MESSAGES: We firstly found that the TERT promoter mutation was strongly associated with malignant SFTs (P = 0.003) and the representative 1b (NAB2ex4-STAT6ex2) or 2a (NAB2ex6-STAT6ex16) fusion variants similarly contribute to tumorigenicity. We also found that TP53 immunopositivity (P = 0.006) and loss of APAF1 immunoreactivity (P < 0.001) were significantly associated with malignant SFTs. Our study suggests that dysfunction of TP53 and APAF1 leads to impaired apoptotic function, and eventually contributes toward malignant SFT transformation.

Cervical Cancer (CC) is a highly aggressive tumor and is one of the leading causes of cancer-related deaths in women. miR-375 was shown to be significantly down-regulated in cervical cancer cells. However, the precise biological functions of miR-375 and the molecular mechanisms underlying its action in CC are largely unknown. miR-375 targets were predicted by bioinformatics target prediction tools and validated using luciferase reporter assay. Herein, we investigated the functional significance of miR-375 and its target gene in CC to identify potential new therapeutic targets. We found that miR-375 expression was significantly downregulated in CC, and astrocyte elevated gene-1 (AEG-1) was identified as a target of miR-375. Our results also showed that ectopic expression of miR-375 suppressed CC cell proliferation, migration, invasion and angiogenesis, and increased the 5-fluorouracil-induced apoptosis and cell cycle arrest in vitro. In contrast, inhibition of miR-375 expression significantly enhanced these functions. Furthermore, HPV - 16 E6/E7 and HPV - 18 E6/E7 significantly down-regulates miR-375 expression in CC. HPV 16/18-E6/E7/miR-375/AEG-1 axis plays an important role in the regulation of cell proliferation, migration, and invasion in CC. Therefore, targeting miR-375/AEG-1 mediated axis could serve as a potential therapeutic target for CC.

Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.

While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase (GalNAc-T14), is highly inversely correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here, we showed that GalNAc-T14 expression was positively associated with the invasive phenotype. Microarray and biochemical analyses revealed that HOXB9, the expression of which was increased in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the β-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of β-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with reduced recurrence-free survival and increased hazard risk, suggesting that targeting β-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC.

Dermatofibrosarcoma protuberans (DFSP) is a very rare soft tissue sarcoma, generally of low-grade malignancy. DFSP is locally aggressive with a high recurrence rate, but metastasis occurs rarely. To investigate the mechanism of metastasis in DFSP, we analyzed the whole exome sequencing data of serial tumor samples obtained from a patient who had a 10-year history of recurrent and metastatic DFSP. Tracking various genomic alterations, namely somatic mutations, copy number variations, and chromosomal rearrangements, we observed a dramatic change in tumor cell population during the occurrence of metastasis in this DFSP case. The new subclone that emerged in metastatic DFSP harbored a completely different set of somatic mutations and new focal amplifications, which had not been observed in the primary clone before metastasis. The COL1A1-PDGFB fusion, characteristic of DFSP, was found in all of the serial samples. Moreover, the break position on the fusion gene was identical in all samples. Based on these observations, we suggest a clonal evolution model to explain the mechanism underlying metastasis in DFSP and identified several candidate target genes responsible for metastatic DFSP by utilizing The Cancer Genome Atlas database. This is the first study to observe clonal evolution in metastatic DFSP and provide insight for a possible therapeutic strategy for imatinib-resistant or metastatic DFSP.

Gastric cancer (GC), one of the most common cancers worldwide, has a high mortality rate due to limited treatment options. Identifying novel and promising molecular targets is a major challenge that must be overcome if treatment of advanced GC is to be successful. Here, we used comparative genomic hybridization and gene expression microarrays to examine genome-wide DNA copy number alterations (CNAs) and global gene expression in 38 GC samples from old and young patients. We identified frequent CNAs, which included copy number gains on chromosomes 3q, 7p, 8q, 20p, and 20q and copy number losses on chromosomes 19p and 21p. The most frequently gained region was 7p21.1 (55%), whereas the most frequently deleted region was 21p11.1 (50%). Recurrent highly amplified regions 17q12 and 7q31.1-7q31.31 harbored two well-known oncogenes: ERBB2 and MET. Correlation analysis of CNAs and gene expression levels identified CAPZA2 (co-amplified with MET) and genes GRB7, MIEN1, PGAP3, and STARD3 (co-amplified with ERBB2) as potential candidate cancer-promoting genes (CPGs). Public dataset analysis confirmed co-amplification of these genes with MET or ERBB2 in GC tissue samples, and revealed that high expression (except for PGAP3) was significantly associated with shorter overall survival. Knockdown of these genes using small interfering RNA led to significant suppression of GC cell proliferation and migration. Reduced GC cell proliferation mediated by CAPZA2 knockdown was attributable to attenuated cell cycle progression and increased apoptosis. This study identified novel candidate CPGs co-amplified with MET or ERBB2, and suggests that they play a functional role in GC.

The receptor tyrosine kinase c-MET regulates processes essential for tissue remodeling and mammalian development. The dysregulation of c-MET signaling plays a role in tumorigenesis. The aberrant activation of c-MET, such as that caused by gene amplification or mutations, is associated with many cancers. c-MET is therefore an attractive therapeutic target, and inhibitors are being tested in clinical trials. However, inappropriate patient selection criteria, such as low amplification or expression level cut-off values, have led to the failure of clinical trials. To include patients who respond to MET inhibitors, the selection criteria must include MET oncogenic addiction. Here, the efficacy of ABN401, a MET inhibitor, was investigated using histopathologic and genetic analyses in MET-addicted cancer cell lines and xenograft models. ABN401 was highly selective for 571 kinases, and it inhibited c-MET activity and its downstream signaling pathway. We performed pharmacokinetic profiling of ABN401 and defined the dose and treatment duration of ABN401 required to inhibit c-MET phosphorylation in xenograft models. The results show that the efficacy of ABN401 is associated with MET status and they highlight the importance of determining the cut-off values. The results suggest that clinical trials need to establish the characteristics of each sample and their correlations with the efficacy of MET inhibitors.