Soft-tissue sarcomas, which result in approximately 10,700 diagnoses and 3,800 deaths per year in the United States, show remarkable histologic diversity, with more than 50 recognized subtypes. However, knowledge of their genomic alterations is limited. We describe an integrative analysis of DNA sequence, copy number and mRNA expression in 207 samples encompassing seven major subtypes. Frequently mutated genes included TP53 (17% of pleomorphic liposarcomas), NF1 (10.5% of myxofibrosarcomas and 8% of pleomorphic liposarcomas) and PIK3CA (18% of myxoid/round-cell liposarcomas, or MRCs). PIK3CA mutations in MRCs were associated with Akt activation and poor clinical outcomes. In myxofibrosarcomas and pleomorphic liposarcomas, we found both point mutations and genomic deletions affecting the tumor suppressor NF1. Finally, we found that short hairpin RNA (shRNA)-based knockdown of several genes amplified in dedifferentiated liposarcoma, including CDK4 and YEATS4, decreased cell proliferation. Our study yields a detailed map of molecular alterations across diverse sarcoma subtypes and suggests potential subtype-specific targets for therapy.

Hyperandrogenism is the primary manifestation of polycystic ovary syndrome (PCOS), which appears to be caused by excess exposure to androgen. As such, androgenized animal models have been developed and investigated to study the etiology of PCOS. Anti-Mullerian hormone (AMH) is known to be associated with follicle growth, and its levels are two to three times higher in women with PCOS than in those with normal ovaries. We studied how duration of androgen administration affects folliculogenesis and AMH expression.

Metastasis is still a major issue in cancer, and the discovery of biomarkers predicting metastatic capacity is essential for the development of better therapeutic strategies for treating lung adenocarcinoma. By using a proteomic approach, we aimed to identify novel predictors for lymph node metastasis in lung adenocarcinoma. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 6 spots differentially expressed between lymph node metastasis-positive and lymph node metastasis-negative groups in a discovery set. Subsequent mass spectrometry showed that 2 of these spots were derived from galectin-4, and western blot analysis confirmed the overexpression of galectin-4 in metastatic samples. The predictive value of galectin-4 was confirmed by immunohistochemical analysis for a validation set consisting of 707 surgically resected specimens of lung adenocarcinomas (stages I to IV). We observed that 148 lung adenocarcinomas (20.9%) expressed galectin-4, which was significantly associated with variables of disease progression such as tumor size (p<0.0001), pleural invasion (p = 0.0071), venous invasion (p = 0.0178), nodal status (p = 0.0007), and TNM stage (p<0.0001). By the multivariate analysis, Galectin-4 expression was revealed as one of the independent predictor for lymph node metastasis, together with solid predominant and micropapillary histologic pattern. Furthermore, galectin-4 expression was revealed to be an independent predictor for lymph node metastasis and an adverse survival factor in patients with lung adenocarcinoma of acinar predominant type. Galectin-4 plays an important role in metastatic process of lung adenocarcinoma. Immunohistochemical testing for galectin-4 expression may be useful together with the detection of specific histology to predict the metastatic potential of lung adenocarcinoma.

Cervical cancer is a major cause of cancer death in females worldwide. Cervical cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are resistant to conventional radiotherapy and chemotherapy, and CSCs/CICs are thought to be responsible for recurrence. Eradication of CSCs/CICs is thus essential to cure cervical cancer. In this study, we isolated cervical CSCs/CICs by sphere culture, and we identified a cancer testis (CT) antigen, CTCFL/BORIS, that is expressed in cervical CSCs/CICs. BORIS has 23 mRNA isoform variants classified by 6 subfamilies (sfs), and they encode 17 different BORIS peptides. BORIS sf1 and sf4 are expressed in both CSCs/CICs and non-CSCs/CICs, whereas BORIS sf6 is expressed only in CSCs/CICs. Overexpression of BORIS sf6 in cervical cancer cells increased sphere formation and tumor-initiating ability compared with those in control cells, whereas overexpression of BORIS sf1 and BORIS sf4 resulted in only slight increases. Thus, BORIS sf6 is a cervical CSC/CIC-specific subfamily and has a role in the maintenance of cervical CSCs/CICs. BORIS sf6 contains a specific c-terminal domain (C34), and we identified a human leukocyte antigen (HLA)-A2-restricted antigenic peptide, BORIS C34_24(9) encoded by BORIS sf6. A BORIS C34_24(9)-specific cytotoxic T cell (CTL) clone showed cytotoxicity for BORIS sf6-overexpressing cervical cancer cells. Furthermore, the CTL clone significantly suppressed sphere formation of CaSki cells. Taken together, the results indicate that the CT antigen BORIS sf6 is specifically expressed in cervical CSCs/CICs, that BORIS sf6 has a role in the maintenance of CSCs/CICs, and that BORIS C34_24(9) peptide is a promising candidate for cervical CSC/CIC-targeting immunotherapy.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as small subpopulation of cancer cells that are endowed with higher tumor-initiating ability. CSCs/CICs are resistant to standard cancer therapies including chemotherapy and radiotherapy, and they are thus thought to be responsible for cancer recurrence and metastasis. Therefore, elucidation of molecular mechanisms of CSCs/CICs is essential to cure cancer. In this study, we analyzed the gene expression profiles of gynecological CSCs/CICs isolated as aldehyde dehydrogenase high (ALDH(high)) cells, and found that MAPK13, PTTG1IP, CAPN1 and UBQLN2 were preferentially expressed in CSCs/CICs. MAPK13 is expressed in uterine, ovary, stomach, colon, liver and kidney cancer tissues at higher levels compared with adjacent normal tissues. MAPK13 gene knockdown using siRNA reduced the ALDH(high) population and abrogated the tumor-initiating ability. These results indicate that MAPK13 is expressed in gynecological CSCs/CICs and has roles in the maintenance of CSCs/CICs and tumor-initiating ability, and MAPK13 might be a novel molecular target for treatment-resistant CSCs/CICs.

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Single cell transcriptome analysis of a cancer tissue can provide objective assessment of subtype population or the activation of each of various microenvironment component cells. In this study, we applied our newly developed technique of single cell analysis to the myometrial infiltration side (M-side) and the endometrial side (E-side) of a human endometrioid adenocarcinoma with squamous differentiation tissues. We also analyzed spherogenic cultures derived from the same tissue to identify putative regulators of stemness in vivo. Cancer cells in the E-side were highly malignant compared with those in the M-side. Many cells on the E-side were positive for spheroid-specific tumorigenesis-related markers including SOX2. In addition, there were higher numbers of epithelial-to-mesenchymal transition (EMT) cells in the E-side compared with the M-side. This study identified a site containing cells with high malignant potential such as EMT and cancer stem-like cells in cancer tissues. Finally, we demonstrate that established endometrioid adenocarcinoma subtype classifiers were variably expressed across individual cells within a tumor. Thus, such intratumoral heterogeneity may be related to prognostic implications.

Hyperandrogenism is one of the cardinal symptoms in polycystic ovary syndrome and plays a key role in the pathogenesis of polycystic ovary syndrome. However, the precise effects and mechanisms of excess androgen during follicular development are still unclear. Here we investigated the effects of androgen on mouse follicle development in vitro. Androgen did not affect the growth of follicles smaller than 160-180 μm in the presence of follicle-stimulating hormone (FSH). However, in the presence of low FSH, androgen supported the growth of follicles larger than 160-180 μm, a size at which growing follicles acquire FSH-dependency. Androgen did not change the mRNA expression of various growth-promoting factors but did increase mRNA expression of the FSH receptor. We suggest that androgen has a positive impact on follicle development by augmentation of the actions of FSH. Therefore, FSH-responsive but FSH-independent follicles grow in the presence of a certain level of FSH or androgen, and androgen compensates for FSH deficiency in FSH-dependent follicles.

Osteosarcoma (OS) is the most common primary malignant bone tumor. However, the therapeutic results of the advanced cases at the first visit were still extremely poor. Therefore, more effective therapeutic options based on molecular profiling of OS are needed. In this study, we investigated the functions of endoplasmic reticulum (ER) stress activities in OS and elucidated whether ER stress inhibitors could exert antitumor effects. The expression of 84 key genes associated with unfolded protein response (UPR) was assessed in four OS cells (143B, MG63, U2OS and KHOS) by RT2 Profiler PCR Arrays. Based on results, we performed both siRNA and inhibitor assays focusing on IRE1α-XBP1 and PERK pathways. All OS cell lines showed resistance to PERK inhibitors. Furthermore, ATF4 and EIF2A inhibition by siRNA did not affect the survival of OS cell lines. On the other hand, IRE1α-XBP1 inhibition by toyocamycin suppressed OS cell growth (IC50: < 0.075 μM) and cell viability was suppressed in all OS cell lines by silencing XBP1 expression. The expression of XBP1s and XBP1u in OS cell lines and OS surgical samples were confirmed using qPCR. In MG63 and U2OS, toyocamycin decreased the expression level of XBP1s induced by tunicamycin. On the other hand, in 143B and KHOS, stimulation by toyocamycin did not clearly change the expression level of XBP1s induced by tunicamycin. However, morphological apoptotic changes and caspase activation were observed in these two cell lines. Inhibition of the IRE1α-XBP1s pathway is expected to be a promising new target for OS.

One cause of the increase in cervical cancer rates in Japan is the long-term stagnation in the cervical cancer screening consultation rate. Therefore, improving the screening consultation rate is of urgent concern to reduce cervical cancer incidence. Self-collected human papilloma virus (HPV) tests have been successfully adopted in several countries, such as the Netherlands and Australia, as a measure of individuals who have not undergone cervical cancer screening in national programs. This study aimed to verify whether self-collected HPV tests presented an effective countermeasure for individuals who had not undergone the recommended cervical cancer screenings.

A limited number of patients with lung squamous cell carcinoma (SCC) benefit clinically from molecular targeted drugs because of a lack of targetable driver alterations. We aimed to understand the prevalence and clinical significance of lysine-specific demethylase 5D (KDM5D) copy number loss in SCC and explore its potential as a predictive biomarker for ataxia-telangiectasia and Rad3-related (ATR) inhibitor treatment. We evaluated KDM5D copy number loss in 173 surgically resected SCCs from male patients using fluorescence in situ hybridization. KDM5D copy number loss was detected in 75 of the 173 patients (43%). Genome-wide expression profiles of the transcription start sites (TSSs) were obtained from 17 SCCs, for which the cap analysis of gene expression assay was performed, revealing that upregulated genes in tumors with the KDM5D copy number loss are associated with 'cell cycle', whereas downregulated genes in tumors with KDM5D copy number loss were associated with 'immune response'. Clinicopathologically, SCCs with KDM5D copy number loss were associated with late pathological stage (p = 0.0085) and high stromal content (p = 0.0254). Multiplexed fluorescent immunohistochemistry showed that the number of tumor-infiltrating CD8+ /T-bet+ T cells was lower in SCCs with KDM5D copy number loss than in wild-type tumors. In conclusion, approximately 40% of the male patients with SCC exhibited KDM5D copy number loss. Tumors in patients who show this distinct phenotype can be 'cold tumors', which are characterized by the paucity of tumor T-cell infiltration and usually do not respond to immunotherapy. Thus, they may be candidates for trials with ATR inhibitors.

Gastrointestinal stromal tumors (GISTs) are typically characterized by activating mutations of the KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Recently, the neurotrophic tyrosine receptor kinase (NTRK) fusion was reported in a small subset of wild-type GIST. We examined trk IHC and NTRK gene expressions in GIST. Pan-trk immunohistochemistry (IHC) was positive in 25 (all 16 duodenal and 9 out of 16 small intestinal GISTs) of 139 cases, and all pan-trk positive cases showed diffuse and strong expression of c-kit. Interestingly, all of these cases showed only trkB but not trkA/trkC expression. Cap analysis of gene expression (CAGE) analysis identified increased number of genes whose promoters were activated in pan-trk/trkB positive GISTs. Imbalanced expression of NTRK2, which suggests the presence of NTRK2 fusion, was not observed in any of trkB positive GISTs, despite higher mRNA expression. TrkB expression was found in duodenal GISTs and more than half of small intestinal GISTs, and this subset of cases showed poor prognosis. However, there was not clear difference in clinical outcomes according to the trkB expression status in small intestinal GISTs. These findings may provide a possible hypothesis for trkB overexpression contributing to the tumorigenesis and aggressive clinical outcome in GISTs of duodenal origin.

Polycystic ovary syndrome (PCOS) is an endocrine disease that is common in women in their reproductive period. Patients with this disease suffer from anovulation and hyperandrogenism. Ovulation induction with exogenous gonadotropin often causes ovarian hyperstimulation syndrome because many small antral follicles pause in their growth. Treatment with insulin sensitizers is reportedly effective for both anovulation associated with PCOS, and suppression of excessive follicular growth; however, the underlying mechanism of action remains unknown. Although pioglitazone is known as an insulin sensitizer, it also has a potent modulator of cell growth and apoptosis irrespective of insulin resistance. To clarify the effect of pioglitazone on follicular growth, we performed in vitro culture of murine preantral follicles. Secondary follicles (100-160 μm in diameter) isolated from 6-week-old ICR mice were individually cultured for 13 days. Culture conditions were as follows: 1) follicle-stimulating hormone (FSH; 33 mIU/mL; control), 2) FSH plus dihydrotestosterone (DHT; 500 ng/mL), 3) FSH plus pioglitazone (5 ng/mL), and 4) FSH plus DHT/pioglitazone. Survival rate and follicle diameter were evaluated, and concentrations of estradiol (E2) and vascular endothelial growth factor (VEGF) in culture media were measured. mRNA expression of various growth-promoting factors and Vegf within follicles were also assessed. Although no significant differences were observed with regard to survival rate, follicle diameters on day 13 were significantly different.Compared with the control group, the DHT group showed enhanced growth, while groups administered pioglitazone showed stagnation of the accelerated growth induced by DHT. Although DHT treatment enhanced the expression of bone morphogenetic protein 2 (Bmp2) mRNA, pioglitazone exposure suppressed induction of Bmp2 mRNA by DHT. Vegf mRNA and protein expression were also significantly reduced when pioglitazone was added to culture media containing DHT.Administration of pioglitazone negatively affected follicular growth and VEGF levels, which may suppress excessive follicular growth and prevent ovarian hyperstimulation syndrome.

Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH(Br)) cells from ovarian cancer cells. Both SP cells and ALDH(Br) cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDH(Low) cells, respectively. We analyzed an SP and ALDH(Br) overlapping population (SP/ALDH(Br)), and the SP/ALDH(Br) population exhibited higher tumor-initiating ability than that of SP cells or ALDH(Br) cells, enabling initiation of tumor with as few as 10(2) cells. Furthermore, SP/ADLH(Br) population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH(Low), MP/ALDH(Br) and MP/ALDH(Low) cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH(Br) population was detected in several gynecological cancer cells with ratios of 0.1% for HEC-1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH(Br) overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.

Colorectal sessile serrated adenoma/polyps (SSA/Ps) are considered early precursor lesions in the serrated neoplasia pathway. Recent studies have shown associations of SSA/Ps with lost MLH1 expression, a CpG island methylator phenotype, and BRAF mutations. However, the molecular biological features of SSA/Ps with early neoplastic progression have not yet been fully elucidated, owing to the rarity of cases of SSA/P with advanced histology such as cytologic dysplasia or invasive carcinoma. In this study, we aimed to elucidate the molecular biological features of SSA/Ps with dysplasia/carcinoma, representing relatively early stages of the serrated neoplasia pathway.

Giant cell tumors of bone (GCTB) are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the protein expression patterns and the results of the network analysis will provide a better understanding of the effects of denosumab administration in patients with GCTB.

A previous proteomics study demonstrated the overexpression of F-actin capping protein subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have high tumorigenicity. Furthermore, CSCs/CICs are resistant to several cancer therapies, and CSCs/CICs are therefore thought to be responsible for cancer recurrence after treatment and distant metastasis. In epithelial ovarian cancer (EOC) cases, disease recurrence after chemotherapy is frequently observed, suggesting ovarian CSCs/CICs are involved. There are four major histological subtypes in EOC, and serous adenocarcinoma and clear cell adenocarcinoma are high-grade malignancies. We therefore analyzed ovarian CSCs/CICs from ovarian carcinoma cell lines (serous adenocarcinoma and clear cell adenocarcinoma) and primary ovarian cancer cells in this study. We isolated ovarian CSCs/CICs as an aldehyde dehydrogenase 1 high (ALDH1(high)) population from 6 EOC cell lines (3 serous adenocarcinomas and 3 clear cell adenocarcinomas) by the ALDEFLUOR assay. ALDH1(high) cells showed greater sphere-forming ability, higher tumorigenicity and greater invasive capability, indicating that ovarian CSCs/CICs are enriched in ALDH1(high) cells. ALDH1(high) cells could also be isolated from 8 of 11 primary ovarian carcinoma samples. Immunohistochemical staining revealed that higher ALDH1 expression levels in ovary cancer cases are related to poorer prognosis in both serous adenocarcinoma cases and clear cell adenocarcinoma cases. Taken together, the results indicate that ALDH1 is a marker for ovarian CSCs/CICs and that the expression level of ALDH1 might be a novel biomarker for prediction of poor prognosis.

Prognostic factors and therapeutic targets are needed for the patients with cervical adenocarcinoma because they have a poor prognosis. Recently, co-expression of multiple receptor tyrosine kinases (RTKs) has been found to be associated with aggressive biological behavior and poor prognosis of several types of malignancy. To evaluate the significance of the expression of multiple RTKs in uterine cervical cancers, we examined the expression profile of RTKs (EGFR, HER2 and c-Met) and the correlation of their expression with clinicopathological features and prognosis of patients with cervical adenocarcinomas. AIS and adenocarcinoma showed strong expression of a single RTK (EGFR, HER2 or c-Met) on the cell membrane in 41 (77.4%) of 53 cases. Twenty (46%) of the 43 adenocarcinoma cases were positive for double or triple RTKs (P = 0.034). Positivity for EGFR and double positivity for EGFR and HER2 (EGFR+/HER2+/c-Met+ and EGFR+/HER2+/c-Met-) were significantly correlated with lymph node metastasis (P = 0.010 for single and P = 0.013 for double) and UICC stage (P = 0.021 for single and P = 0.007 for double). Positivity for HER2 was significantly correlated with tumor size (P = 0.029). Relapse-free survival (RFS) was significantly shorter in patients who were double positive for EGFR and HER2. Our results suggest that EGFR and HER2 are potential therapeutic targets and that their co-expression is a prognostic factor for cervical adenocarcinoma.

Bone marrow-derived mesenchymal stem cells (BM-MSC) has been applied as the most valuable source of autologous cell transplantation for various diseases including diabetic complications. However, hyperglycemia may cause abnormalities in intrinsic BM-MSC which might lose sufficient therapeutic effects in diabetic patients. We demonstrated the functional abnormalities in BM-MSC derived from both type 1 and type 2 diabetes models in vitro, which resulted in loss of therapeutic effects in vivo in diabetic nephropathy (DN). Then, we developed a novel method to improve abnormalities in BM-MSC using human umbilical cord extracts, namely Wharton's jelly extract supernatant (WJs). WJs is a cocktail of growth factors, extracellular matrixes and exosomes, which ameliorates proliferative capacity, motility, mitochondrial degeneration, endoplasmic reticular functions and exosome secretions in both type 1 and type 2 diabetes-derived BM-MSC (DM-MSC). Exosomes contained in WJs were a key factor for this activation, which exerted similar effects to complete WJs. DM-MSC activated by WJs ameliorated renal injury in both type 1 and type 2 DN. In this study, we developed a novel activating method using WJs to significantly increase the therapeutic effect of BM-MSC, which may allow effective autologous cell transplantation.