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Mechanisms regulating the gene expression program at different hypoxia severity levels in patient tumors are not understood. We aimed to determine microRNA (miRNA) regulation of this program at defined hypoxia levels from moderate to severe in prostate cancer. Biopsies from 95 patients were used, where 83 patients received the hypoxia marker pimonidazole before prostatectomy. Forty hypoxia levels were extracted from pimonidazole-stained histological sections and correlated with miRNA and gene expression profiles determined by RNA sequencing and Illumina bead arrays. This identified miRNAs associated with moderate (n = 7) and severe (n = 28) hypoxia and predicted their target genes. The scores of miRNAs or target genes showed prognostic significance, as validated in an external cohort of 417 patients. The target genes showed enrichment of gene sets for cell proliferation and MYC activation at all hypoxia levels and PTEN inactivation at severe hypoxia. This was confirmed by RT-qPCR for MYC and PTEN, by Ki67 immunohistochemistry, and by gene set analysis in an external cohort. To assess whether miRNA regulation occurred within the predicted hypoxic regions, a method to quantify co-localization of multiple histopathology parameters at defined hypoxia levels was applied. A high Ki67 proliferation index co-localized significantly with hypoxia at all levels. The co-localization index was strongly associated with poor prognosis. Absence of PTEN staining co-localized significantly with severe hypoxia. The scores for miRNAs correlated with the co-localization index for Ki67 staining and hypoxia, consistent with miRNA regulation within the overlapping regions. This was confirmed by showing miR-210-3p expression within severe hypoxia by in situ hybridization. Cell line experiments (22Rv1, PC3) were conducted to determine whether miRNAs and target genes were regulated directly by hypoxia. Most of them were hypoxia-unresponsive, and probably regulated by other mechanisms such as MYC activation. In conclusion, in aggressive, hypoxic prostate tumors, cancer cells exhibit different proliferative gene expression programs that is regulated by miRNAs and depend on whether the cells reside in moderate or severe hypoxic regions. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Cervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend human papilloma virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy., Results: Marker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections (N = 252) and cervical smears (N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% [95% CI 74.12-100.00], and specificity of 91% [95% CI 62.26-99.53] to 96% [95% CI 79.01-99.78], and receiver operating characteristic area under the curve (ROC AUC) = 1.000 [95% CI 1.00-1.00] compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% [95% CI 37.77-70.84] to 89% [95% CI 67.20-98.03], specificity of 93% [95% CI 84.07-97.38] to 96% [95% CI 79.01-99.78], and a ROC AUC ranging from 0.793 [95% CI 0.68-0.89] to 0.99 [95% CI 0.97-1.00] compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% [95% CI 77.45-92.69], specificity 95% [95% CI 88.64-98.18], and ROC AUC = 0.925 [95% CI 0.878-0.974] compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11-80.44), specificity of 94% (95% CI 88.30-97.40), and ROC AUC = 0.884 (95% CI 0.822-0.945) compared to low-grade intraepithelial lesion (LSIL)/normal in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers., Conclusions: This 5-marker panel detected SCC and HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted., (© 2024. The Author(s).)

Purpose: To develop an immune-based gene expression risk score to identify patients with cervical cancer at increased risk of distant metastases (DM)., Experimental Design: Tumor biopsies were obtained from 81 patients prior to chemoradiotherapy. Whole-transcriptome RNA sequencing was performed (Illumina NextSeq500). Beginning with 4,723 immune-related genes, a 55-gene risk score for DM was derived using Cox modeling and principal component analysis. It was validated in independent cohorts of 274 patients treated at the Norwegian Radium Hospital (NRH) and 206 patients from The Cancer Genome Atlas (TCGA)., Results: The risk score was predictive of DM (HR, 2.7; P < 0.0001) and lower cause-specific survival (CSS) by univariate analysis (HR, 2.0; P = 0.0003) and multivariate analysis adjusted for clinical factors (DM HR, 3.0; P < 0.0001; CSS HR, 2.2; P = 0.0004). The risk score predicted DM (HR, 1.4; P = 0.05) and CSS (HR, 1.48; P = 0.013) in the NRH cohort and CSS (HR, 1.4; P = 0.03) in TCGA cohort. Higher risk scores were associated with lower CIBERSORT estimates of tumor-infiltrating immune cells, including CD8 T cells and M1 and M2 macrophages (all P < 0.001). Higher risk scores were associated with lower expression (all P < 0.001) of important chemokines (CXCL12, CXCR4), IFN-regulated genes (IRF1, STAT1, IDO1), and immune checkpoint regulators (PD-1, PD-L1, CTLA-4)., Conclusions: The immune metastatic risk score addresses important challenges in the treatment of cervical cancer-identifying patients at high risk of DM after radiotherapy. The findings of this study indicate that high tumor mutational burden and a "cold," immune-excluded tumor microenvironment influence distant metastatic recurrence. Further validation of the risk score is needed., (©2024 American Association for Cancer Research.)

Rationale: Current therapies for metastatic osseous disease frequently fail to provide a durable treatment response. To date, there are only limited therapeutic options for metastatic prostate cancer, the mechanisms that drive the survival of metastasis-initiating cells are poorly characterized, and reliable prognostic markers are missing. A high aldehyde dehydrogenase (ALDH) activity has been long considered a marker of cancer stem cells (CSC). Our study characterized a differential role of ALDH1A1 and ALDH1A3 genes as regulators of prostate cancer progression and metastatic growth. Methods: By genetic silencing of ALDH1A1 and ALDH1A3 in vitro , in xenografted zebrafish and murine models, and by comparative immunohistochemical analyses of benign, primary tumor, and metastatic specimens from patients with prostate cancer, we demonstrated that ALDH1A1 and ALDH1A3 maintain the CSC phenotype and radioresistance and regulate bone metastasis-initiating cells. We have validated ALDH1A1 and ALDH1A3 as potential biomarkers of clinical outcomes in the independent cohorts of patients with PCa. Furthermore, by RNAseq, chromatin immunoprecipitation (ChIP), and biostatistics analyses, we suggested the molecular mechanisms explaining the role of ALDH1A1 in PCa progression. Results: We found that aldehyde dehydrogenase protein ALDH1A1 positively regulates tumor cell survival in circulation, extravasation, and metastatic dissemination, whereas ALDH1A3 plays the opposite role. ALDH1A1 and ALDH1A3 are differentially expressed in metastatic tumors of patients with prostate cancer, and their expression levels oppositely correlate with clinical outcomes. Prostate cancer progression is associated with the increasing interplay of ALDH1A1 with androgen receptor (AR) and retinoid receptor (RAR) transcriptional programs. Polo-like kinase 3 (PLK3) was identified as a transcriptional target oppositely regulated by ALDH1A1 and ALDH1A3 genes in RAR and AR-dependent manner. PLK3 contributes to the control of prostate cancer cell proliferation, migration, DNA repair, and radioresistance. ALDH1A1 gain in prostate cancer bone metastases is associated with high PLK3 expression. Conclusion: This report provides the first evidence that ALDH1A1 and PLK3 could serve as biomarkers to predict metastatic dissemination and radiotherapy resistance in patients with prostate cancer and could be potential therapeutic targets to eliminate metastasis-initiating and radioresistant tumor cell populations., Competing Interests: Competing Interests: In the past 5 years, Dr. Mechthild Krause received funding for her research projects by IBA (2016), Merck KGaA (2014-2018 for preclinical study; 2018-2020 for clinical study), Medipan GmbH (2014-2018). In the past 5 years, Dr. Krause, Dr. Linge and Dr. Löck have been involved in an ongoing publicly funded (German Federal Ministry of Education and Research) project with the companies Medipan, Attomol GmbH, GA Generic Assays GmbH, Gesellschaft für medizinische und wissenschaftliche genetische Analysen, Lipotype GmbH and PolyAn GmbH (2019-2021). For the present manuscript, none of the above-mentioned funding sources were involved., (© The author(s).)

Background and Purpose: The biology behind individual hypoxia levels in patient tumors is poorly understood. Here, we used radiogenomics to identify associations between magnetic resonance imaging (MRI)-based hypoxia levels and biological processes derived from gene expression data in prostate cancer., Materials and Methods: For 85 prostate cancer patients, MRI-based hypoxia images were constructed by combining diffusion-weighted images reflecting oxygen consumption and supply. The ability to differentiate hypoxia levels in these images was verified by comparison with matched biopsy sections stained for the hypoxia marker pimonidazole. For MRI-defined hypoxia levels, corresponding hypoxic fractions were calculated and correlated with biopsy gene expression profiles. Biological processes were predicted by gene set enrichment analysis (GSEA) and validated by immunohistochemistry (Ki67 proliferation marker, reactive stroma grade) and RT-PCR (MYC)., Results: Genes with correlation between expression level and hypoxic fraction were identified for 56 MRI-based hypoxia levels. At all levels, GSEA identified proliferation as the predominant biological process enriched among the correlating genes. Two independent proliferative gene signatures were developed. The Peak1 signature, upregulated at moderate/severe hypoxia, reflected MYC upregulation and high Ki67-proliferation index of cancer cells in pimonidazole-positive regions. The Peak2 signature, upregulated at mild to non-hypoxic levels, was associated with fibroblast gene signature and reactive stroma grade. High scores of both Peak1 and Peak2 indicated elevated risk of biochemical recurrence in multiple cohorts., Conclusion: Radiogenomics identified two gene expression programs activated at different hypoxia levels, reflecting proliferation of cancer cells and stroma cells. Genes involved in these programs could be candidate targets for intervention., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Human papillomavirus (HPV)-associated cervical cancer is a leading cause of cancer deaths in women. Here we present an integrated multi-omic analysis of 643 cervical squamous cell carcinomas (CSCC, the most common histological variant of cervical cancer), representing patient populations from the USA, Europe and Sub-Saharan Africa and identify two CSCC subtypes (C1 and C2) with differing prognosis. C1 and C2 tumours can be driven by either of the two most common HPV types in cervical cancer (16 and 18) and while HPV16 and HPV18 are overrepresented among C1 and C2 tumours respectively, the prognostic difference between groups is not due to HPV type. C2 tumours, which comprise approximately 20% of CSCCs across these cohorts, display distinct genomic alterations, including loss or mutation of the STK11 tumour suppressor gene, increased expression of several immune checkpoint genes and differences in the tumour immune microenvironment that may explain the shorter survival associated with this group. In conclusion, we identify two therapy-relevant CSCC subtypes that share the same defining characteristics across three geographically diverse cohorts., (© 2022. The Author(s).)

Background: Gene signatures measured in a biopsy have been proposed as hypoxia biomarkers in prostate cancer. We assessed a previously developed signature, and aimed to determine its relationship to hypoxia and its heterogeneity within the dominant (index) lesion of prostate cancer., Methods: The 32-gene signature was assessed from gene expression data of 141 biopsies from the index lesion of 94 patients treated with prostatectomy. A gene score calculated from the expression levels was applied in the analyses. Hypoxic fraction from pimonidazole immunostained whole-mount and biopsy sections was used as reference standard for hypoxia., Results: The gene score was correlated with pimonidazole-defined hypoxic fraction in whole-mount sections, and the two parameters showed almost equal association with clinical markers of tumour aggressiveness. Based on the gene score, incorrect classification according to hypoxic fraction in whole-mount sections was seen in one third of the patients. The incorrect classifications were apparently not due to intra-tumour heterogeneity, since the score had low heterogeneity compared to pimonidazole-defined hypoxic fraction in biopsies. The score showed prognostic significance in uni-and multivariate analysis in independent cohorts., Conclusions: Our signature from the index lesion reflects tumour hypoxia and predicts prognosis in prostate cancer, independent of intra-tumour heterogeneity in pimonidazole-defined hypoxia., (© 2022. The Author(s).)

Many patients with locally advanced cervical cancer experience recurrence within the radiation field after chemoradiotherapy. Biomarkers of tumor radioresistance are required to identify patients in need of intensified treatment. Here, the biomarker potential of miR-200 family members was investigated in this disease. Also, involvement of tumor hypoxia in the radioresistance mechanism was determined, using a previously defined 6-gene hypoxia classifier. miR-200 expression was measured in pretreatment tumor biopsies of an explorative cohort (n = 90) and validation cohort 1 (n = 110) by RNA sequencing. Publicly available miR-200 data of 79 patients were included for the validation of prognostic significance. A score based on expression of the miR-200a/b/-429 (miR-200a, miR-200b, and miR-429) cluster showed prognostic significance in all cohorts. The score was significant in multivariate analysis of central pelvic recurrence. No association with distant recurrence or hypoxia status was found. Potential miRNA target genes were identified from gene expression profiles and showed enrichment of genes in extracellular matrix organization and cell adhesion. miR-200a/b/-429 overexpression had a pronounced radiosensitizing effect in tumor xenografts, whereas the effect was minor in vitro. In conclusion, miR-200a/b/-429 downregulation is a candidate biomarker of central pelvic recurrence and seems to predict cell adhesion-mediated tumor radioresistance independent of clinical markers and hypoxia., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Hypoxia arises in tumor regions with insufficient oxygen supply and is a major barrier in cancer treatment. The distribution of hypoxia levels is highly heterogeneous, ranging from mild, almost non-hypoxic, to severe and anoxic levels. The individual hypoxia levels induce a variety of biological responses that impair the treatment effect. A stronger focus on hypoxia levels rather than the absence or presence of hypoxia in our investigations will help development of improved strategies to treat patients with hypoxic tumors. Current knowledge on how hypoxia levels are sensed by cancer cells and mediate cellular responses that promote treatment resistance is comprehensive. Recently, it has become evident that hypoxia also has an important, more unexplored role in the interaction between cancer cells, stroma and immune cells, influencing the composition and structure of the tumor microenvironment. Establishment of how such processes depend on the hypoxia level requires more advanced tumor models and methodology. In this review, we describe promising model systems and tools for investigations of hypoxia levels in tumors. We further present current knowledge and emerging research on cellular responses to individual levels, and discuss their impact in novel therapeutic approaches to overcome the hypoxia barrier.

Tumor hypoxia levels range from mild to severe and have different biological and therapeutical consequences but are not easily assessable in patients. Here we present a method based on diagnostic dynamic contrast enhanced (DCE) MRI that reflects a continuous range of hypoxia levels in patients with tumors of cervical cancer. Hypoxia images were generated using an established approach based on pixel-wise combination of DCE-MRI parameters ν e and K trans , representing oxygen consumption and supply, respectively. Using two tumor models, an algorithm to retrieve surrogate measures of hypoxia levels from the images was developed and validated by comparing the MRI-defined levels with hypoxia levels reflected in pimonidazole-stained histologic sections. An additional indicator of hypoxia levels in patient tumors was established on the basis of expression of nine hypoxia-responsive genes; a strong correlation was found between these indicator values and MRI-defined hypoxia levels in 63 patients. Chemoradiotherapy outcome of 74 patients was most strongly predicted by moderate hypoxia levels, whereas more severe or milder levels were less predictive. By combining gene expression profiles and MRI-defined hypoxia levels in cancer hallmark analysis, we identified a distribution of levels associated with each hallmark; oxidative phosphorylation and G 2 -M checkpoint were associated with moderate hypoxia, epithelial-to-mesenchymal transition, and inflammatory responses with significantly more severe levels. At the mildest levels, IFN response hallmarks together with HIF1A protein expression by IHC appeared significant. Thus, our method visualizes the distribution of hypoxia levels within patient tumors and has potential to distinguish levels of different prognostic and biological significance. SIGNIFICANCE: These findings present an approach to image a continuous range of hypoxia levels in tumors and demonstrate the combination of imaging with molecular data to better understand the biology behind these different levels., (©2020 American Association for Cancer Research.)

Background: Emerging biomarkers from medical imaging or molecular characterization of tumour biopsies open up for combining the two and exploiting their synergy in treatment planning of cancer patients. We generated a paired data set of imaging- and gene-based hypoxia biomarkers in cervical cancer, appraised the influence of intratumour heterogeneity in patient classification, and investigated the benefit of combining the methodologies in prediction of chemoradiotherapy failure., Methods: Hypoxic fraction from dynamic contrast enhanced (DCE)-MR images and an expression signature of six hypoxia-responsive genes were assessed as imaging- and gene-based biomarker, respectively in 118 patients., Findings: Dichotomous biomarker cutoff to yield similar hypoxia status by imaging and genes was defined in 41 patients, and the association was validated in the remaining 77 patients. The two biomarkers classified 75% of 118 patients with the same hypoxia status, and inconsistent classification was not related to imaging-defined intratumour heterogeneity in hypoxia. Gene-based hypoxia was independent on tumour cell fraction in the biopsies and showed minor heterogeneity across multiple samples in 9 tumours. Combining imaging- and gene-based classification gave a significantly better prediction of PFS than one biomarker alone. A combined dichotomous biomarker optimized in 77 patients showed a large separation in PFS between more and less hypoxic tumours, and separated the remaining 41 patients with different PFS. The combined biomarker showed prognostic value together with tumour stage in multivariate analysis., Interpretation: Combining imaging- and gene-based biomarkers may enable more precise and informative assessment of hypoxia-related chemoradiotherapy resistance in cervical cancer., Funding: Norwegian Cancer Society, South-Eastern Norway Regional Health Authority, and Norwegian Research Council., Competing Interests: Declaration of Competing Interest HL is registered as inventor of a patent application covering the clinical use of the hypoxia gene signature (WO2013/124,738)., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

CDK regulatory subunit 2 (CKS2) has a nuclear function that promotes cell division and is a candidate biomarker of chemoradioresistance in cervical cancer. The underlying mechanisms are, however, not completely understood. We investigated whether CKS2 also has a mitochondrial function that augments tumor aggressiveness. Based on global gene expression data of two cervical cancer cohorts of 150 and 135 patients, we identified a set of genes correlated with CKS2 expression. Gene set enrichment analysis showed enrichment of mitochondrial cellular compartments, and the hallmarks oxidative phosphorylation (OXPHOS) and targets of the MYC oncogene in the gene set. By in situ proximity ligation assay, we showed that CKS2 formed complex with the positively correlated MYC target, mitochondrial single-stranded DNA binding protein SSBP1, in the mitochondrion of cervix tumor samples and HeLa and SiHa cervical cancer cell lines, indicating a role in mitochondrial DNA (mtDNA) replication and thereby OXPHOS. CDK1 was found to be part of the complex. Flow cytometry analyses of HeLa cells showed cell cycle regulation of the CKS2-SSBP1 complex consistent with mtDNA replication activity. Moreover, repression of mtDNA replication and OXPHOS by acute hypoxia decreased CKS2-SSBP1 complex abundance and expression of MYC targets. By immunohistochemistry, cytoplasmic CKS2 expression was found to add to the prognostic impact of nuclear CKS2 expression in patients, suggesting that the mitochondrial function promotes tumor aggressiveness. Our study uncovers a novel link between regulation of cell division by nuclear pathways and OXPHOS in the mitochondrion that involves CKS2 and promotes chemoradioresistance of cervical cancer., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: A 31-gene expression signature reflected in dynamic contrast enhanced (DCE)-MR images and correlated with hypoxia-related aggressiveness in cervical cancer was identified in previous work. We here aimed to construct a dichotomous classifier with key signature genes and a predefined classification threshold that separated cervical cancer patients into a more and less hypoxic group with different outcome to chemoradiotherapy., Experimental Design: A training cohort of 42 patients and two independent cohorts of 108 and 131 patients were included. Gene expression data were generated from tumor biopsies by two Illumina array generations (WG-6, HT-12). Technical transfer of the classifier to a reverse transcription quantitative PCR (RT-qPCR) platform was performed for 74 patients. The amplitude ABrix in the Brix pharmacokinetic model was extracted from DCE-MR images of 64 patients and used as an indicator of hypoxia., Results: Classifier candidates were constructed by integrative analysis of ABrix and gene expression profiles in the training cohort and evaluated by a leave-one-out cross-validation approach. On the basis of their ability to separate patients correctly according to hypoxia status, a 6-gene classifier was identified. The classifier separated the patients into two groups with different progression-free survival probability. The robustness of the classifier was demonstrated by successful validation of hypoxia association and prognostic value across cohorts, array generations, and assay platforms. The prognostic value was independent of existing clinical markers, regardless of clinical endpoints., Conclusions: A robust DCE-MRI-associated gene classifier has been constructed that may be used to achieve an early indication of patients' risk of hypoxia-related chemoradiotherapy failure. Clin Cancer Res; 22(16); 4067-76. ©2016 AACR., (©2016 American Association for Cancer Research.)

Hypoxia is an adverse factor in cervical cancer, and hypoxia-related gene expression could be a powerful biomarker for identifying the aggressive hypoxic tumors. Reverse transcription quantitative PCR (RT-qPCR) is a valuable method for gene expression studies, but suitable reference genes for data normalization that are independent of hypoxia status and clinical parameters of cervical tumors are lacking. In the present work, we aimed to identify reference genes for RT-qPCR studies of hypoxia in squamous cervical cancer. From 422 candidate reference genes selected from the literature, we used Illumina array-based expression profiles to identify 182 genes not affected by hypoxia in cervical cancer, i.e. genes regulated by hypoxia in eight cervical cancer cell lines or correlating with the hypoxia-associated dynamic contrast-enhanced magnetic resonance imaging parameter ABrix in 42 patients, were excluded. Among the 182 genes, nine candidates (CHCHD1, GNB2L1, IPO8, LASP1, RPL27A, RPS12, SOD1, SRSF9, TMBIM6) that were not associated with tumor volume, stage, lymph node involvement or disease progression in array data of 150 patients, were selected for further testing by RT-qPCR. geNorm and NormFinder analyses of RT-qPCR data of 74 patients identified CHCHD1, SRSF9 and TMBIM6 as the optimal set of reference genes, with stable expression both overall and across patient subgroups with different hypoxia status (ABrix) and clinical parameters. The suitability of the three reference genes were validated in studies of the hypoxia-induced genes DDIT3, ERO1A, and STC2. After normalization, the RT-qPCR data of these genes showed a significant correlation with Illumina expression (P<0.001, n = 74) and ABrix (P<0.05, n = 32), and the STC2 data were associated with clinical outcome, in accordance with the Illumina data. Thus, CHCHD1, SRSF9 and TMBIM6 seem to be suitable reference genes for studying hypoxia-related gene expression in squamous cervical cancer samples by RT-qPCR. Moreover, STC2 is a promising prognostic hypoxia biomarker in cervical cancer.

Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved prognostic markers.

Background: How cells decipher the duration of an external signal into different transcriptional outcomes is poorly understood. The hormone gastrin can promote a variety of cellular responses including proliferation, differentiation, migration and anti-apoptosis. While gastrin in normal concentrations has important physiological functions in the gastrointestine, prolonged high levels of gastrin (hypergastrinemia) is related to pathophysiological processes., Results: We have used genome-wide microarray time series analysis and molecular studies to identify genes that are affected by the duration of gastrin treatment in adenocarcinoma cells. Among 403 genes differentially regulated in transiently (gastrin removed after 1 h) versus sustained (gastrin present for 14 h) treated cells, 259 genes upregulated by sustained gastrin treatment compared to untreated controls were expressed at lower levels in the transient mode. The difference was subtle for early genes like Junb and c-Fos, but substantial for delayed and late genes. Inhibition of protein synthesis by cycloheximide was used to distinguish between primary and secondary gastrin regulated genes. The majority of gastrin upregulated genes lower expressed in transiently treated cells were primary genes induced independently of de novo protein synthesis. This indicates that the duration effect of gastrin treatment is mainly mediated via post-translational signalling events, while a smaller fraction of the differentially expressed genes are regulated downstream of primary transcriptional events. Indeed, sustained gastrin treatment specifically induced prolonged ERK1/2 activation and elevated levels of the AP-1 subunit protein JUNB. Enrichment analyses of the differentially expressed genes suggested that endoplasmic reticulum (ER) stress and survival is affected by the duration of gastrin treatment. Sustained treatment exerted an anti-apoptotic effect on serum starvation-induced apoptosis via a PKC-dependent mechanism. In accordance with this, only sustained treatment induced anti-apoptotic genes like Clu, Selm and Mcl1, while the pro-apoptotic gene Casp2 was more highly expressed in transiently treated cells. Knockdown studies showed that JUNB is involved in sustained gastrin induced expression of the UPR/ER stress related genes Atf4, Herpud1 and Chac1., Conclusion: The duration of gastrin treatment affects both intracellular signalling mechanisms and gene expression, and ERK1/2 and AP-1 seem to play a role in converting different durations of gastrin treatment into distinct cellular responses.

The peptide hormone gastrin is an important factor for the maintenance and homeostasis of the gastric mucosa. We show that gastrin stimulates proliferation in a dose-dependent manner in the human gastric adenocarcinoma cell line AGS-GR. Furthermore, we demonstrate that the MAPK scaffold protein MEK partner 1 (MP1) is important for gastrin-induced phosphorylation of ERK1 and ERK2 and that MP1 promotes gastrin-induced proliferation of AGS-GR cells. Our results suggest a role of MP1 in gastrin-induced cellular responses involved in proliferation and homeostasis of the gastric mucosa.

We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.

RNA-transfected cell microarray shows great promise in functional genomics. By printing siRNA complexed with transfection reagent on glass slides, arrays of transfected cells are formed in which the phenotypic consequences of gene suppression can be investigated. Using reporter plasmids with fluorescence intensity as output data, we have developed a strategy for statistical analysis of the intensity data from medium-scale functional studies using data from several experimental replicates.

Transfected cell microarray is a promising method for accelerating the functional exploration of the genome, giving information about protein function in the living cell. The microarrays consist of clusters of cells (spots) overexpressing or silencing a particular gene product. The subsequent analysis of the phenotypic consequences of such perturbations can then be detected using cell-based assays. The focus in the present study was to establish an experimental design and a robust analysis approach for fluorescence intensity data, and to address the use of replicates for studying regulation of gene expression with varying complexity and effect size. Our analysis pipeline includes measurement of fluorescence intensities, normalization strategies using negative control spots and internal control plasmids, and linear regression (ANOVA) modelling for estimating biological effects and calculating P-values for comparisons of interests. Our results show the potential of transfected cell microarrays in studying complex regulation of gene expression by enabling measurement of biological responses in cells with overexpression and downregulation of specific gene products, combined with the possibility of assaying the effects of external stimuli. Simulation experiments show that transfected cell microarrays can be used to reliably detect even quantitatively minor biological effects by including several technical and experimental replicates.