PURPOSE: Glioblastoma is still intractable despite the progress in therapies, and the intractability is attributable to a minor population of stem-like tumor cells. As a niche harboring quiescent stem-like tumor cells with potentially high tumorigenicity, we have specified an area around large ischemic necrosis, termed 'peri-necrotic niche', in glioblastoma. In this study, the behavior of tumor cells inside and outside the peri-necrotic niche was analyzed to find out molecules responsible for reactivation of quiescent stem-like tumor cells to proliferate outside the niche.
METHODS: Expression of Ki-67 and GINS complex composed of SLD5, PSF1, PSF2 and PSF3 was analyzed by immunohistochemistry in human glioblastoma tissue samples. Proliferation assays, immunoblotting and siRNA experiments were performed using a glioblastoma cell line.
RESULTS: Immunohistochemical analysis revealed quiescent and proliferative phenotypes of tumor cells inside and outside the niche, respectively, and the proliferation was spatially correlated with the expression of GINS components in tumor cells. To mimic the tissue microenvironment inside versus outside the niche, glioblastoma cells were cultured under hypoxic versus normoxic conditions, or without versus with serum. Quiescence and proliferation of tumor cells were reversibly determined by the microenvironment inside and outside the niche, respectively, and proliferative activities paralleled the expression levels of GINS components. Furthermore, the reactivation of proliferation after reoxygenation or serum replenishment was suppressed in quiescent tumor cells with PSF1 knockdown.
CONCLUSIONS: These findings indicate the essential role of GINS complex in the switch between quiescence and proliferation of tumor cells inside and outside the peri-necrotic niche.

Lysophospholipase1 (LYPLA1) also known as acyl‑protein thioesterase1 (APT1) belongs to the superfamily of α/β hydrolase. It has been found to have the properties of a homodimer by manifesting depalmitoylation as well as lysophospholipase activity. LYPLAs are under the control of both microRNAs, miR‑138 and miR‑424. They were observed to be significantly overexpressed in chronic lymphocytic leukemia cells. To date, LYPLAs are the sole enzymes recognized to activate depalmitoylation. In this study, we provide the expression pattern of LYPLA1 in non‑small cell lung cancer (NSCLC) using four different NSCLC cell lines. Western blot analysis and RT‑PCR were performed to detect the protein expression and mRNA expression of LYPLA1 in NSCLC cell lines. We detected the highest LYPLA1 protein expression level in SPC‑A‑1 cells followed by A549 cells, and the highest LYPLA1 mRNA expression level was detected in the SPC‑A‑1 cells followed by the H1299 cell line. We found that suppression of LYPLA1 expression using small‑interfering RNA significantly inhibited proliferation, migration and invasion of the LYPLA1‑transfected NSCLC cells. Furthermore, we explored the involvement of LYPLA1 in the regulation of epithelial‑mesenchymal transition (EMT). The epithelial marker E‑cadherin was significantly increased, while mesenchymal markers N‑cadherin, vimentin and SNAIL were markedly decreased in the LYPLA1‑silenced cells. Collectively the results of the present study suggest that the LYPLA1 gene plays a tumor‑promotor role in NSCLC cells in vitro.

BACKGROUND & AIMS: Immune checkpoint inhibition may affect growth or progression of highly aggressive cancers, such as esophageal adenocarcinoma (EAC). We investigated the regulation of expression of major histocompatibility complex, class 1 (MHC-I) proteins (encoded by HLA-A, HLA-B, and HLA-C) and the immune response to EACs in patient samples.
METHODS: We performed quantitative polymerase chain reaction array analyses of OE33 cells and OE19 cells, which express different levels of the ATP binding cassette subfamily B member 1 (TAP1) and TAP2, required for antigen presentation by MHC-I, to identify microRNAs (miRNAs) that regulate their expression. We performed luciferase assays to validate interactions between miRNAs and potential targets. We overexpressed candidate miRNAs in OE33, FLO-1, and OACP4 C cell lines and performed quantitative polymerase chain reaction, immunoblot, and flow cytometry analyses to identify changes in messenger RNA (mRNA) and protein expression; we studied the effects of cytotoxic T cells. We performed miRNA in situ hybridization, RNA-sequencing, and immunohistochemical analyses of tumor tissues from 51 untreated patients with EAC in the Netherlands. Clinical and survival data were collected for patients, and EAC subtypes were determined.
RESULTS: We found OE19 cells to have increased levels of 7 miRNAs. Of these, we found binding sites for miRNA 125a (MIR125a)-5p in the 3' untranslated region of the TAP2 mRNA and binding sites for MIR148a-3p in 3' untranslated regions of HLA-A, HLA-B, and HLA-C mRNAs. Overexpression of these miRNAs reduced expression of TAP2 in OE33, FLO-1, and OACP4 C cells, and reduced cell-surface levels of MHC-I. OE33 cells that expressed the viral peptide BZLF1 were killed by cytotoxic T cells, whereas OE33 that overexpressed MIR125a-5p or MIR 148a along with BZLF1 were not. In EAC and nontumor tissues, levels of MIR125a-5p correlated inversely with levels of TAP2 protein. High expression of TAP1 by EAC correlated with significantly shorter overall survival times of patients. EACs that expressed high levels of TAP1 and genes involved in antigen presentation also expressed high levels of genes that regulate the adaptive immune response, PD-L1, PD-L2, and IDO1; these EACs had a poor response to neoadjuvant chemoradiotherapy and associated with shorter overall survival times of patients.
CONCLUSIONS: In studies of EAC cell lines and tumor tissues, we found increased levels of MIR125a-5p and MIR148a-3p to reduce levels of TAP2 and MHC-I, required for antigen presentation. High expression of MHC-I molecules by EAC correlated with markers of an adaptive immune response and significantly shorter overall survival times of patients.

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor

Asymmetric cell division results in two distinctly fated daughter cells. A molecular hallmark of asymmetric division is the unequal partitioning of cell fate determinants. We have previously established that growth factor signaling promotes protein depalmitoylation to foster polarized protein localization, which, in turn, drives migration and metastasis. We report protein palmitoylation as a key mechanism for the asymmetric partitioning of the cell fate determinants Numb and β-catenin through the activity of the depalmitoylating enzyme APT1. Using point mutations, we showed that specific palmitoylated residues on Numb were required for its asymmetric localization. By live-cell imaging, we showed that reciprocal interactions between APT1 and the Rho family GTPase CDC42 promoted the asymmetric localization of Numb and β-catenin to the plasma membrane. This, in turn, restricted Notch- or Wnt-responsive transcriptional activity to one daughter cell. Moreover, we showed that altering APT1 abundance changed the transcriptional signatures of MDA-MB-231 triple receptor-negative breast cancer cells, similar to changes in Notch and β-catenin-mediated Wnt signaling. We also showed that loss of APT1 depleted a specific subpopulation of tumorigenic cells in colony formation assays. Together, our findings suggest that APT1-mediated depalmitoylation is a major mechanism of asymmetric cell division that maintains Notch- and Wnt-associated protein dynamics, gene expression, and cellular functions.

In this study, we describe a liposome-based siRNA delivery system with a core composed of siRNA:protamine complex and a shell designed for the active targeting of CD44-expressing cells using for the first time the anti-CD44 aptamer (named Apt1) as targeting ligand. Among all functions, CD44 is the most common cancer stem cell surface biomarker and is found overexpressed in many tumors making this an attractive receptor for therapeutic targeting. This unique non-cationic system was evaluated for the silencing of the reporter gene of luciferase (luc2) in a triple-negative breast cancer model in vitro and in vivo. We show the possibility of conjugating an aptamer to siRNA-containing liposomes for an efficient gene silencing in CD44-expressing tumor cells in vivo, in the perspective of silencing disease-related genes in tumors.

The Kazakh population in Xinjiang Province in northwestern China exhibits a high incidence of esophageal squamous cell carcinoma (ESCC). Although the etiology of esophageal carcinoma (EC) has not been elucidated, there are reports of the involvement of an immunologic mechanism. In the current study, 268 Kazakh ESCC patients and 500 age- and sex-matched control subjects were recruited. DNA was extracted from paraffin-embedded tumor specimens from the patients and peripheral blood lymphocytes from the controls and used for LMP2/LMP7 genotyping. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed to detect LMP2/LMP7 gene single-nucleotide polymorphisms (SNPs). We found a clear increased risk of ESCC in the Kazakh population for the heterozygous LMP2 R/C genotype and the homozygous C/C genotype (OR = 1.470, 95%CI = 1.076-2.008, p = 0.015 forLMP2R/C; OR = 2.048, 95% CI = 1.168-3.591, p = 0.011 for LMP2 C/C). Conversely, the heterozygous LMP7 Q/K polymorphism was found to decrease the risk of ESCC in this population (OR = 0.421, 95% CI = 0.286-0.621, p = 8.83×10-6). Moreover, LMP2 R/C+C/C genotype was associated with increased tumor invasion depth (p = 0.041). Haplotype analysis showed that haplotype A, which includes wild-type homozygous LMP2/TAP1 and mutant LMP7, decreases susceptibility to ESCC in the Kazakh population; in contrast, haplotype E, which includes wild-type homozygous LMP2/LMP7/TAP1, acts as a risk factor for increased susceptibility to ESCC. This is the first study to report that the heterozygous LMP2 R/C and homozygous C/C genotypes increase susceptibility to ESCC in the Kazakh population and that the heterozygous LMP7 Q/K genotype decreases susceptibility to ESCC in this population. Nevertheless, neither LMP2 nor LMP7 was associated with human papillomavirus (HPV) infection. Understanding LMP2/LMP7 genetic variability will provide a new therapeutic perspective for Kazakh patients with ESCC.

BACKGROUND: Recently, immunotherapy with anti-PD-1 antibodies has shown clinical benefit in recurrent Small Cell Lung Cancer (SCLC). Since anti-PD-1 re-activates anti-tumor Cytotoxic T Lymphocyte (CTL) responses, it is crucial to understand the mechanisms regulating HLA class I, and PD-L1 expression in HLA-negative SCLC. Here we addressed the role of IL-27, a cytokine related to both IL-6 and IL-12 families.
METHODS: The human SCLC cell lines NCI-N592, -H69, -H146, -H446 and -H82 were treated in vitro with different cytokines (IL-27, IFN-γ, IL-6 or a soluble IL-6R/IL-6 chimera [sIL-6R/IL-6]) at different time points and analyzed for tyrosine-phosphorylated STAT proteins by Western blot, for surface molecule expression by immunofluorescence and FACS analyses or for specific mRNA expression by QRT-PCR. Relative quantification of mRNAs was calculated by the ΔΔCT method. The Student's T test was used for the statistical analysis of experimental replicates.
RESULTS: IL-27 triggered STAT1/3 phosphorylation and up-regulated the expression of surface HLA class I antigen and of TAP1 and TAP2 mRNA in four out of five SCLC cell lines tested. The IL-27-resistant NCI-H146 cells showed up-regulation of HLA class I by IFN-γ. IFN-γ also induced expression of PD-L1 in SCLC cells, while IL-27 was less potent in this respect. IL-27 failed to activate STAT1/3 phosphorylation in NCI-H146 cells, which display a low expression of the IL-27RA and GP130 receptor chains. As GP130 is shared in IL-27R and IL-6R complexes, we assessed its functionality in response to sIL-6R/IL-6. sIL-6R/IL-6 failed to trigger STAT1/3 signaling in NCI-H146 cells, suggesting low GP130 expression or uncoupling from signal transduction. Although both sIL-6R/IL-6 and IL-27 triggered STAT1/3 phosphorylation, sIL-6R/IL-6 failed to up-regulate HLA class I expression, in relationship to the weak activation of STAT1. Finally sIL-6R/IL-6 limited IL-27-effects, particularly in NCI-H69 cells, in a SOCS3-independent manner, but did not modify IFN-γ induced HLA class I up-regulation.
CONCLUSIONS: In conclusion, IL-27 is a potentially interesting cytokine for restoring HLA class I expression for SCLC combined immunotherapy purposes. However, the concomitant activation of the IL-6 pathway may limit the IL-27 effect on HLA class I induction but did not significantly alter the responsiveness to IFN-γ.

Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer. The latter is due to its viral or UV-associated carcinogenesis. For tumor progression MCC has to escape the host's immuno-surveillance, e.g. by loss of HLA class-I expression. Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines. This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2. Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells. Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components. Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model. In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins. In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.

Estrogen receptor alpha (ERα) also known as NR3A1 (nuclear receptor subfamily 3, group A, member 1) is a ligand-activated transcription factor. It is an important biomarker for breast cancer metastasis. In the present study, we report a novel DNA aptamer candidate against estrogen receptor (ER) alpha structure. The enriched aptamer candidate was obtained after 14 iterative cycles of in vitro protein-SELEX process. Isothermal calorimetry study suggests the nanomolar sensitivity of the candidate ER_Apt1 to its target protein. Fluorescence- and chemiluminescence-binding assays confirm the specificity of the candidate aptamer to ER alpha positive breast cancer cell line. Comparative analysis of ER_Apt1 to ER alpha monoclonal antibody was also performed to analyze the expression of ER alpha in various malignant cancer cell line. Cytochemical and immunohistochemistry assay indicates its potential use as a diagnostic agent against ERα positive carcinomas. The nucleotide aptamer sequences described in the present study can be used for the detection, treatment, prophylaxis and diagnosis of ERα-related disorder.

INTRODUCTION: Uveal melanoma (UM) with an inflammatory phenotype, characterized by infiltrating leukocytes and increased human leukocyte antigen (HLA) expression, carry an increased risk of death due to metastases. These tumors should be ideal for T-cell based therapies, yet it is not clear why prognostically-infaust tumors have a high HLA expression. We set out to determine whether the level of HLA molecules in UM is associated with other genetic factors, HLA transcriptional regulators, or microenvironmental factors.
METHODS: 28 enucleated UM were used to study HLA class I and II expression, and several regulators of HLA by immunohistochemistry, PCR microarray, qPCR and chromosome SNP-array. Fresh tumor samples of eight primary UM and four metastases were compared to their corresponding xenograft in SCID mice, using a PCR microarray and SNP array.
RESULTS: Increased expression levels of HLA class I and II showed no dosage effect of chromosome 6p, but, as expected, were associated with monosomy of chromosome 3. Increased HLA class I and II protein levels were positively associated with their gene expression and with raised levels of the peptide-loading gene TAP1, and HLA transcriptional regulators IRF1, IRF8, CIITA, and NLRC5, revealing a higher transcriptional activity in prognostically-bad tumors. Implantation of fresh human tumor samples into SCID mice led to a loss of infiltrating leukocytes, and to a decreased expression of HLA class I and II genes, and their regulators.
CONCLUSION: Our data provides evidence for a proper functioning HLA regulatory system in UM, offering a target for T-cell based therapies.

BACKGROUND: Novel therapies for men with castration-resistant prostate cancer (CRPC) are needed, particularly for cancers not driven by androgen receptor (AR) activation.
OBJECTIVES: To identify molecular subgroups of PC bone metastases of relevance for therapy.
DESIGN, SETTING, AND PARTICIPANTS: Fresh-frozen bone metastasis samples from men with CRPC (n=40), treatment-naïve PC (n=8), or other malignancies (n=12) were characterized using whole-genome expression profiling, multivariate principal component analysis (PCA), and functional enrichment analysis. Expression profiles were verified by reverse transcription-polymerase chain reaction (RT-PCR) in an extended set of bone metastases (n=77) and compared to levels in malignant and adjacent benign prostate tissue from patients with localized disease (n=12). Selected proteins were evaluated using immunohistochemistry. A cohort of PC patients (n=284) diagnosed at transurethral resection with long follow-up was used for prognostic evaluation.
RESULTS AND LIMITATIONS: The majority of CRPC bone metastases (80%) was defined as AR-driven based on PCA analysis and high expression of the AR, AR co-regulators (FOXA1, HOXB13), and AR-regulated genes (KLK2, KLK3, NKX3.1, STEAP2, TMPRSS2); 20% were non-AR-driven. Functional enrichment analysis indicated high metabolic activity and low immune responses in AR-driven metastases. Accordingly, infiltration of CD3
CONCLUSIONS: Most CRPC bone metastases show high AR and metabolic activities and low immune responses. A subgroup instead shows low AR and metabolic activities, but high immune responses. Targeted therapy for these groups should be explored.
PATIENT SUMMARY: We studied heterogeneities at a molecular level in bone metastasis samples obtained from men with castration-resistant prostate cancer. We found differences of possible importance for therapy selection in individual patients.

PURPOSE: Cetuximab, an EGFR-specific antibody (mAb), modestly improves clinical outcome in patients with head and neck cancer (HNC). Cetuximab mediates natural killer (NK) cell:dendritic cell (DC) cross-talk by cross-linking FcγRIIIa, which is important for inducing antitumor cellular immunity. Cetuximab-activated NK cells upregulate the costimulatory receptor CD137 (4-1BB), which, when triggered by agonistic mAb urelumab, might enhance NK-cell functions, to promote T-cell-based immunity.
EXPERIMENTAL DESIGN: CD137 expression on tumor-infiltrating lymphocytes was evaluated in a prospective cetuximab neoadjuvant trial, and CD137 stimulation was evaluated in a phase Ib trial, in combining agonistic urelumab with cetuximab. Flow cytometry and cytokine release assays using NK cells and DC were used in vitro, testing the addition of urelumab to cetuximab-activated NK, DC, and cross presentation to T cells.
RESULTS: CD137 agonist mAb urelumab enhanced cetuximab-activated NK-cell survival, DC maturation, and tumor antigen cross-presentation. Urelumab boosted DC maturation markers, CD86 and HLA DR, and antigen-processing machinery (APM) components TAP1/2, leading to increased tumor antigen cross-presentation. In neoadjuvant cetuximab-treated patients with HNC, upregulation of CD137 by intratumoral, cetuximab-activated NK cells correlated with FcγRIIIa V/F polymorphism and predicted clinical response. Moreover, immune biomarker modulation was observed in an open label, phase Ib clinical trial, of patients with HNC treated with cetuximab plus urelumab.
CONCLUSIONS: These results suggest a beneficial effect of combination immunotherapy using cetuximab and CD137 agonist in HNC. Clin Cancer Res; 23(3); 707-16. ©2016 AACR.

BACKGROUND: PSF1 (Partner of SLD Five 1) is an evolutionarily conserved DNA replication factor that is part of the GINS (Go, Ichi, Nii, and San) complex . The objective of this study was to evaluate the relationship between PSF1 expression and prognosis in patients with non-small cell lung cancer (NSCLC) treated with surgery following preoperative chemotherapy or chemoradiotherapy.
METHODS: Sixty-nine patients with NSCLC treated with surgery following preoperative chemotherapy or chemoradiotherapy who did not achieve pathologic complete response were enrolled. The status of PSF1 expression was evaluated by immunohistochemistry, and the relationship between expression of PSF1 and Ki-67 was determined, as well as correlations between PSF1 expression and prognosis.
RESULTS: We found that 27 of 69 patients' tumors (39 %) were positive for PSF1 expression. The Ki-67 index was significantly higher in the PSF1-positive versus the PSF1-negative group (p = 0.0026). Five-year, disease-free survival of the PSF1-positive group was significantly worse (17.7 vs. 44.3 %, p = 0.0088), and the 5-year overall survival also was worse (16.6 vs. 47.2 %, p = 0.0059). Moreover, PSF1 expression was found to be a significant independent prognostic factor for shorter survival by Cox multivariate analysis (hazard ratio 2.43, 95 % confidence interval 1.27-4.60, p = 0.0076).
CONCLUSIONS: PSF1 is a useful prognostic biomarker to stratify NSCLC patients treated with surgery following preoperative chemotherapy or chemoradiotherapy.

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.

Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

It has been reported that the presence of a small group of cancer stem‑like 'side population (SP)' cells is responsible for therapy failure and tumor recurrence. The present study demonstrated that primary human osteosarcoma samples contained a SP of about 3.9% which overexpressed ABC transporters, including ABCA1, ABCB1, ABCB2 and ABCG2, which are associated with drug resistance and may have contributed to multi‑drug resistance of SP cells. Furthermore, these SP cells displayed increased expression of endosialin (CD248) and other stem cell surface proteins, including CD133, octamer‑binding transcription factor 3/4A, Nanog and Nestin, which are ultimately responsible for high self‑renewal and deregulated cell proliferation. In addition, it was shown that endosialin‑overexpressing SP cells were able to regenerate the tumor population and had a high invasive potential. Therefore, the present study suggested that osteosarcoma SP cells were cancer stem cells, as they displayed stem‑like properties; furthermore, endosialin may be a potential target to prevent osteosarcoma recurrence following chemotherapy.

OBJECTIVE: To validate our earlier observation that 11 chemoresistance-associated mRNAs are molecular markers of poor overall survival in ovarian serous carcinoma.
METHODS: Ovarian serous carcinomas (n=112) and solid metastases (n=63; total=175) were analyzed for mRNA expression of APC, BAG3, EGFR, S100A10, ITGAE, MAPK3, TAP1, BNIP3, MMP9, FASLG and GPX3 using quantitative real-time PCR. mRNA expression was studied for association with clinicopathologic parameters and survival. Tumor heterogeneity was assessed in 20 cases with >1 specimen per patient. APC, BAG3, S100A10 and ERK1 protein expression by immunohistochemistry was analyzed in 58 specimens (38 primary carcinomas, 20 metastases).
RESULTS: BAG3 (p=0.013), TAP1 (p=0.014), BNIP3 (p<0.001) and MMP9 (p=0.036) were overexpressed in primary tumors, whereas S100A10 (p=0.027) and FASLG (p=0.006) were overexpressed in metastases. Analysis of patient-matched primary carcinomas and metastases showed overexpression of APC (p=0.022), MAPK3 (p=0.002) and BNIP3 (p=0.004) in the former. In primary carcinomas, higher APC (p=0.003) and MAPK3 (p=0.005) levels were related to less favorable chemoresponse. Higher S100A10 (p=0.029) and MAPK3 (p=0.041) levels were related to primary chemoresistance. Higher BAG3 (p=0.026) and APC (p=0.046) levels in primary carcinomas were significantly related to poor overall survival in univariate, though not in multivariate survival analysis. S100A10 protein expression was related to poor chemoresponse (p=0.002) and shorter overall (p=0.005) and progression-free (p<0.001) survival, the latter finding retained in multivariate analysis (p=0.035).
CONCLUSIONS: Our data provide evidence of heterogeneity in ovarian serous carcinoma and identify APC, MAPK3, BAG3 and S100A10 as potential biomarkers of poor chemotherapy response and/or poor outcome in this cancer.

BACKGROUND: The role of human papillomavirus (HPV) may be involved in the development of esophageal cancer (EC) and the polymorphic immune response gene transporter associated with antigen processing (TAP) may be involved in HPV persistence and subsequent cancer carcinogenesis. The current study aims to provide association evidence for HPV with EC, to investigate TAP1 polymorphisms in EC and assess its association with HPV statuses and EC in Kazakhs.
METHODS: The HPV genotypes in 361 patients with EC and 66 controls selected from Kazakh population were evaluated using PCR. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to detect two SNPs of TAP1 in 150 cases comprised of 75 HPV(+) and 75 HPV(-) patients and 283 pure ethnic population of Kazakh and evaluate their associations with susceptibility to EC. A case-to-case comparison based on the genotyping results was conducted to address the function of TAP1 variants in the involvement of HPV.
RESULTS: The presence of four HPV genotypes in EC tissues - including HPV 16, 18, 31, 45 - was significantly higher at 64.6 % than those in controls at 18.2 % (P < 0.001). Such presence was strongly associated with increased risk of EC (OR 8.196; 95 % CI 4.280-15.964). The infection of HPV16, and multi-infection of 16 and 18 significantly increase the risk for developing EC (OR 4.616, 95 % CI 2.099-10.151; and OR 6.029, 95 % CI 1.395-26.057 respectively). Heterozygote of TAP1 D637G had a significantly higher risk for developing EC (OR 1.626; 95 % CI 1.080-2.449). The odds ratio for HPV infection was significantly lower among carriers of TAP1 D637G polymorphism (OR 0.281; 95 % CI 0.144-0.551).
CONCLUSIONS: HPV infection exhibits a strong positive association with the risk of EC in Kazakhs. Heterozygote of TAP1 D637G decreases susceptibility to HPV infection in patients with EC but increases susceptibility to EC among the Kazakh populations.

Kinase suppressor of Ras 1 (KSR1) has been implicated in tumorigenesis in multiple cancers, including skin, pancreatic and lung carcinomas. However, our recent study revealed a role of KSR1 as a tumour suppressor in breast cancer, the expression of which is potentially correlated with chemotherapy response. Here, we aimed to further elucidate the KSR1-regulated signalling in response to genotoxic agents in breast cancer. Stable isotope labelling by amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS) was implemented to globally characterise cellular protein levels induced by KSR1 in the presence of doxorubicin or etoposide. The acquired proteomic signature was compared and GO-STRING analysis was subsequently performed to illustrate the activated functional signalling networks. Furthermore, the clinical associations of KSR1 with identified targets and their relevance in chemotherapy response were examined in breast cancer patients. We reveal a comprehensive repertoire of thousands of proteins identified in each dataset and compare the unique proteomic profiles as well as functional connections modulated by KSR1 after doxorubicin (Doxo-KSR1) or etoposide (Etop-KSR1) stimulus. From the up-regulated top hits, several proteins, including STAT1, ISG15 and TAP1 are also found to be positively associated with KSR1 expression in patient samples. Moreover, high KSR1 expression, as well as high abundance of these proteins, is correlated with better survival in breast cancer patients who underwent chemotherapy. In aggregate, our data exemplify a broad functional network conferred by KSR1 with genotoxic agents and highlight its implication in predicting chemotherapy response in breast cancer.

The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies, and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was used to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2-dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1(-/-) cells. Cetuximab enhanced HNC cell recognition by EGFR853-861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271-279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity.

Genetic variation of antigen-processing machinery (APM) components has been shown to be associated with cervical carcinoma risk and outcome in a genetically homogeneous Dutch population. However, the role of APM component single nucleotide polymorphisms (SNPs) in genetically heterogeneous populations with different distributions of human papillomavirus (HPV) subtypes remains unclear. Eleven non-synonymous, coding SNPs in the TAP1, TAP2, LMP2, LMP7 and ERAP1 genes were genotyped in cervical carcinoma patients and healthy controls from two distinct Indonesian populations (Balinese and Javanese). Individual genotype and allele distributions were investigated using single-marker analysis, and combined SNP effects were assessed by haplotype construction and haplotype interaction analysis. Allele distribution patterns in Bali and Java differed in relation to cervical carcinoma risk, with four ERAP1 SNPs and one TAP2 SNP in the Javanese population showing significant association with cervical carcinoma risk, while in the Balinese population, only one TAP2 SNP showed this association. Multimarker analysis demonstrated that in the Javanese patients, one specific haplotype, consisting of the ERAP1-575 locus on chromosome 5 and the TAP2-379 and TAP2-651 loci on chromosome 6, was significantly associated with cervical carcinoma risk (global P = 0.008); no significant haplotype associations were found in the Balinese population. These data indicate not only that genetic variation in APM component genes is associated with cervical carcinoma risk in Indonesia but also that the patterns of association differ depending on background genetic composition and possibly on differences in HPV type distribution.

Development of colorectal cancer (CRC) may result from a dysfunctional interplay between diet, gut microbes and the immune system. The ABC transport proteins ABCB1 (P-glycoprotein, Multidrug resistance protein 1, MDR1), ABCC2 (MRP2) and ABCG2 (BCRP) are involved in transport of various compounds across the epithelial barrier. Low mRNA level of ABCB1 has previously been identified as an early event in colorectal carcinogenesis (Andersen et al., PLoS One. 2013 Aug 19;8(8):e72119). ABCC2 and ABCG2 mRNA levels were assessed in intestinal tissue from 122 CRC cases, 106 adenoma cases (12 with severe dysplasia, 94 with mild-moderate dysplasia) and from 18 controls with normal endoscopy. We found significantly higher level of ABCC2 in adenomas with mild to moderate dysplasia and carcinoma tissue compared to the levels in unaffected tissue from the same individual (P = 0.037, P = 0.037, and P<0.0001) and in carcinoma and distant unaffected tissue from CRC cases compared to the level in the healthy individuals (P = 0.0046 and P = 0.036). Furthermore, ABCG2 mRNA levels were significantly lower in adenomas and carcinomas compared to the level in unaffected tissue from the same individuals and compared to tissue from healthy individuals (P<0.0001 for all). The level of ABCB2 in adjacent normal tissue was significantly higher than in tissue from healthy individuals (P = 0.011). In conclusion, this study found that ABCC2 and ABCG2 expression levels were altered already in mild/moderate dysplasia in carcinogenesis suggesting that these ABC transporters are involved in the early steps of carcinogenesis as previously reported for ABCB1. These results suggest that dysfunctional transport across the epithelial barrier may contribute to colorectal carcinogenesis.

Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) with PSF-TFE3 gene fusion is a rare neoplasm. Only 22 cases of Xp11.2 RCCs with PSF-TFE3 have been reported to date. We describe an additional case of Xp11.2 RCC with PSF-TFE3 showing melanotic features. Microscopically, the histologic features mimic clear cell renal cell carcinoma. However, the dark-brown pigments were identified and could be demonstrated as melanins. Immunohistochemically, the tumor cells were widely positive for CD10, human melanoma black 45, and TFE3 but negative for cytokeratins, vimentin, Melan-A, microphthalmia-associated transcription factor, smooth muscle actin, and S-100 protein. Genetically, we demonstrated PSF-TFE3 fusion between exon 9 of PSF and exon 5 of TFE3. The patient was free of disease with 50 months of follow-up. The prognosis of this type of tumor requires more cases because of limited number of cases and follow-up period. Xp11.2 RCC with PSF-TFE3 inevitably requires differentiation from other kidney neoplasms. Immunohistochemical and molecular genetic analyses are essential for accurate diagnosis.

BACKGROUND: Partner of Sld five 3 (PSF3) is a member of the evolutionarily conserved heterotetrameric complex "Go-Ichi-Ni-San" (GINS), which consists of SLD5, PSF1, PSF2, and PSF3. Previous studies have suggested that some GINS complex members are upregulated in cancer, but the status of PSF3 expression in colorectal cancer has not been investigated.
METHODS: We investigated the status of PSF3 expression in 137 consecutive resected colorectal caners by quantitative reverse-transcription polymerase chain reaction. Univariable and multivariable Cox regression analyses were performed to assess independent prognostic factors for overall survival in colorectal cancer.
RESULTS: In 137 restected colorectal cancer samples, median messenger RNA (mRNA) expression levels of PSF3 were significantly higher in tumor tissues (1.35 × 10(-3), range 2.88 × 10(-4) to 3.16 × 10(-2)) than in adjacent normal tissues (2.94 × 10(-4), range 5.48 × 10(-5) to 1.27 × 10(-3)) (P < 0.05). Moreover, high expression of PSF3 in tumor tissues was associated with shorter disease-free survival and overall survival. When analyzed with a Cox regression model, the PSF3 expression was an independent prognostic factor for overall survival. In addition, in patients with early stage (stage I and II) colorectal cancer, the overall survival rate of the high PSF3 expression group was significantly lower than that of the low PSF3 expression group (P < 0.001).
CONCLUSIONS: The PSF3 expression plays an important role in the progression of colorectal cancer and acts as a factor significantly affecting the prognosis of patients.
VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_217.

BACKGROUND: Partner of SLD5 1 (PSF1) is an evolutionarily conserved DNA replication factor. Previous studies have suggested that transcriptional activity of the PSF1 gene correlated with malignancy of cancer cells. The objective of the current study was to evaluate the relationship between PSF1 expression and the clinical features of prostate cancer.
METHODS: We determined the expression of PSF1 in 120 needle biopsy samples of prostate cancer by immunohistochemistry. We divided patients into PSF1-positive or -negative groups and analyzed the relationships between the expression of PSF1, the Gleason score, PSA level, TNM classification and prognosis.
RESULTS: Our results showed that the PSF1 expression correlated significantly with PSA values at diagnosis (P=0.0028), with tumor grade (P<0.0001), and with clinical stage (P=0.0005). Moreover, the PSF1 expression correlated significantly with overall survival (hazard ratio (HR) 5.5; 95% confidence interval (CI) 2.17-15.8; P=0.003) and progression-free survival in 99 consecutive patients with prostate cancer. Noteworthy, the prognosis of PSF1-positive cases was also worse in patients with a Gleason score of 8-10 (HR 3.7; 95% CI 1.28-13.43; P=0.0143). Limitations include that this study had a retrospective design, that patients in the study were heterogeneous and included those with early and advanced cancer, and that small tumor fragments may not be representative of the entire carcinoma.
CONCLUSIONS: PSF1 is expressed in high-grade prostate cancer and may be a useful biomarker to identify patients with a poor prognosis at the time of diagnosis.

Partner of sld five 1 (PSF1) is a member of the heterotetrameric complex termed GINS. Previous studies have shown that PSF1 is unregulated in several cancer and associated with tumor malignant characters. However, the effects of PSF1 in lung cancer are still unclear. The goal of this study was to investigate the effects of PSF1 on the proliferation capacities of lung cancer. To start with, expression of PSF1 in 22 human lung cancer samples and adjacent non-tumor samples were detected by real-time RT-PCR and Western blotting. Our results showed that PSF1 was overexpressed in lung cancer samples compared to adjacent non-tumor samples. To achieve better insights of PSF1 functions in lung cancer cells, we used PSF1-specific small interfering RNA (siRNA) successfully inhibit the expression of PSF1 in messenger RNA (mRNA) and protein levels. In addition, we used lung cancer cell lines with different p53 gene background (p53 null and p53 wild-type). The results showed that knockdown of PSF1 inhibited cell proliferation and caused cell cycle arrest of lung cancer cells in a p53-independent manner. Our data indicated that PSF1 is functionally involved in lung cancer cell proliferation and is a potential target for lung cancer therapy.

BACKGROUND: PSF1 is a subunit of the GINS complex which is essential for establishment of DNA replication forks, and the progression of the replisome. Previous studies have shown a close relationship between PSF1 and cell cycle in the proliferation of immature cells as well as tumors. The purpose of this study was to measure PSF1 expression in hepatocellular carcinoma (HCC) tissues, and determine the effects of down-regulation of PSF1 expression on growth of cancer cells, the cell cycle, apoptosis and cell invasiveness.
METHODS: Samples from 137 HCC tissues, 67 from adjacent nontumor tissue and 15 from normal liver were studied using immunochemistry. The HepG2 cell line was used for knockdown experiments studied by RT-PCR, real-time PCR, apoptosis and invasiveness assays.
RESULTS: PSF1 was overexpressed in HCC tissues compared with normal liver tissues. High PSF1 expression correlated with a more aggressive phenotype as well as worse prognosis in HCC patients. Knockdown of PSF1 expression using small interfering RNA (siRNA) slowed the growth of cancer cell by suppressing the cell cycle progression as well as increasing apoptosis, especially early apoptosis. In addition, the invasiveness of HepG2 cells was also reduced by down-regulation of PSF1.
CONCLUSIONS: These results suggest that the inhibition of PSF1 might provide new therapeutic approaches for HCC.

The major histocompatibility complex (MHC) region on chromosome 6p21.3 is suspected to host susceptibility loci for HIV-related Kaposi's sarcoma (HIV-KS). A nested case-control study in the Multicenter AIDS Cohort Study was designed to conduct fine genetic association mapping across central MHC. Individuals co-infected with HIV-1 and human herpes virus-8 who later developed KS were defined as cases (n=354) and were matched 1:1 with co-infected KS-free controls. We report data for new independent MHC class II and III susceptibility loci. In particular, class II HLA-DMB emerged as a strong candidate, with the intronic variant rs6902982 A>G associated with a fourfold increase of risk (odds ratio (OR)=4.09; 95% confidence interval (CI)=1.90-8.80; P=0.0003). A striking multiplicative effect on the estimated risk was associated with further carriage of two non-synonymous variants, rs1800453 A>G (Asp697Gly) and rs4148880 A>G (Ile393Val), in the linked TAP1 gene (OR=10.5; 95% CI=2.54-43.6; P=0.0012). The class III susceptibility variant is moderately associated with HIV-KS and lies within a 120-kb-long haplotype (OR=1.52; 95% CI=1.01-2.28; P=0.047) formed by rs7029 A>G (GPANK1 3' untranslated region), rs1065356 G>A (LY6G6C), rs3749953 A>G (MSH5-SAPCD1 read through) and rs707926 G>A (VARS). Our data suggest that antigen processing by MHC class II molecules is a target pathway in the pathogenesis of HIV-KS.