In vivo chemoresistance of prostate cancer in metronomic cyclophosphamide therapy

A human prostate cancer (PC3) xenograft model was established which reflects acquired in vivo resistance towards metronomic cyclophosphamide (CPA) treatment. Cell cultures of two in vivo resistant PC3 tumors were established which maintain chemoresistant phenotypes upon xenografting into mice. A comparative proteome analysis of the two resistant cell lines PC3-D3 and -D4 versus the non-resistant parental PC3 cell line by 2D-DIGE approach followed by MALDI-TOF–TOF analysis revealed a total of 25 differently expressed proteins. Validation of protein candidates by Western blot analysis of the corresponding in vivo tumor xenografts identified three differentially expressed proteins (thioredoxin containing protein 5, cathepsin B, and annexin A3). Thioredoxin containing protein 5 was up-regulated in resistant xenografts only upon in vivo CPA therapy. A truncated version of cathepsin B translocated into mitochondria in the resistant clones whereas it stays cytoplasmic in corresponding parental PC3 cells. Annexin A3 (ANXA3) presents a very interesting candidate which was found to be up-regulated both in vitro and in xenografts, with protein levels further increased by metronomic CPA treatment in vivo. It is noteworthy that independent studies in other epithelial cancers recently identified ANXA3 as cancer progression and resistance marker.     

A new set of differentially expressed signaling genes is early expressed in coffee leaf rust race II incompatible interaction

New races of coffee rust are overcoming resistance genes available in germplasm and cultivated cultivars and bringing recently some coffee-producing countries in severe economic challenge. The objective of this study was to identify the genes that are linked to host resistance to the major coffee rust race II. In our study, we have identified and studied a segregating population that has a single monogenic resistant gene to coffee rust. Coffee leaves of parents, resistant, and susceptible genotypes of the F₂ generation plants were inoculated with pathogen spores. A differential analysis was performed by combined cDNA-AFLP and bulk segregant analysis (BSA) in pooled samples collected 48 and 72 h postinoculation, increasing the selectiveness for differential gene expression. Of 108 differential expressed genes, between 33,000 gene fragments analyzed, 108 differential expressed genes were identified in resistant plants. About 20 and 22 % of these resistant-correlated genes are related to signaling and defense genes, respectively. Between signaling genes, the major subclass corresponds to receptor and resistant homolog genes, like nucleotide-binding site leucine-rich repeat (NBS-LRR), Pto-like, RLKs, Bger, and RGH1A, all not previously described in coffee rust responses. The second major subclass included kinases, where two mitogen-activated kinases (MAPK) are identified. Further gene expression analysis was performed for 21 selected genes by real-time PCR gene expression analysis at 0, 12, 24, 48, and 72 h postinoculation. The expression of genes involved in signaling and defense was higher at 24 and 72 h after inoculation, respectively. The NBS-LRR was the more differentially expressed gene between the signaling genes (four times more expressed in the resistant genotype), and thraumatin (PR5) was the more expressed between all genes (six times more expressed). Multivariate analysis reinforces the significance of the temporal separation of identified signaling and defense genes: early expression of signaling genes support the hypothesis that higher expression of the signaling components up regulates the defense genes identified. Additionally the increased gene expression of these two gene sets is associated with a single monogenic resistance trait to to leaf coffee rust in the interaction characterized here.     

TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often develop drug resistance, but mechanisms linking thrombin and hypoxia to drug resistance remain unresolved. Our studies using Doxorubicin (DOX) resistant MCF7 breast cancer cells reveals a mechanism linking DOX exposure with hypoxic induction of DOX resistance. Global expression changes between parental and DOX resistant MCF7 cells were examined. Westerns, Northerns and immunocytochemistry were used to validate drug resistance and differentially expressed genes. A cluster of genes involved in the anticoagulation pathway, with Tissue Factor Pathway Inhibitor 1 (TFPI1) the top hit, was identified. Plasmids overexpressing TFPI1 were utilized, and 1% O₂ was used to test the effects of hypoxia on drug resistance. Lastly, microarray datasets from patients with drug resistant breast tumors were interrogated for TFPI1 expression levels. TFPI1 protein levels were found elevated in 3 additional DOX resistant cells lines, from humans and rats, indicating evolutionarily conservation of the effect. Elevated TFPI1 in DOX resistant cells was active, as thrombin protein levels were coincidentally low. We observed elevated HIF1α protein in DOX resistant cells, and in cells with forced expression of TFPI1, suggesting TFPI1 induces HIF1α. TFPI1 also induced c-MYC, c-SRC, and HDAC2 protein, as well as DOX resistance in parental cells. Growth of cells in 1% O₂ induced elevated HIF1α, BCRP and MDR-1 protein, and these cells were resistant to DOX. Our in vitro results were consistent with in vivo patient datasets, as tumors harboring increased BCRP and MDR-1 expression also had increased TFPI1 expression. Our observations are clinically relevant indicating that DOX treatment induces an anticoagulation cascade, leading to inhibition of thrombin and the expression of HIF1α. This in turn activates a pathway leading to drug resistance.     

Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors

The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition.     

Identifying Resistance Mechanisms against Five Tyrosine Kinase Inhibitors Targeting the ERBB/RAS Pathway in 45 Cancer Cell Lines

Because of the low overall response rates of 10–47% to targeted cancer therapeutics, there is an increasing need for predictive biomarkers. We aimed to identify genes predicting response to five already approved tyrosine kinase inhibitors. We tested 45 cancer cell lines for sensitivity to sunitinib, erlotinib, lapatinib, sorafenib and gefitinib at the clinically administered doses. A resistance matrix was determined, and gene expression profiles of the subsets of resistant vs. sensitive cell lines were compared. Triplicate gene expression signatures were obtained from the caArray project. Significance analysis of microarrays and rank products were applied for feature selection. Ninety-five genes were also measured by RT-PCR. In case of four sunitinib resistance associated genes, the results were validated in clinical samples by immunohistochemistry. A list of 63 top genes associated with resistance against the five tyrosine kinase inhibitors was identified. Quantitative RT-PCR analysis confirmed 45 of 63 genes identified by microarray analysis. Only two genes (ANXA3 and RAB25) were related to sensitivity against more than three inhibitors. The immunohistochemical analysis of sunitinib-treated metastatic renal cell carcinomas confirmed the correlation between RAB17, LGALS8, and EPCAM and overall survival. In summary, we determined predictive biomarkers for five tyrosine kinase inhibitors, and validated sunitinib resistance biomarkers by immunohistochemistry in an independent patient cohort.     

Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer

Exosomes are carriers of intercellular information that regulate the tumor microenvironment, and they have an essential role in drug resistance through various mechanisms such as transporting RNA molecules and proteins. Nevertheless, their effects on gemcitabine resistance in triple-negative breast cancer (TNBC) are unclear. In the present study, we examined the effects of exosomes on TNBC cell viability, colony formation, apoptosis, and annexin A6 (ANXA6)/EGFR expression. We addressed their roles in gemcitabine resistance and the underlying mechanism. Our results revealed that exosomes derived from resistant cancer cells improved cell viability and colony formation and inhibited apoptosis in sensitive cancer cells. The underlying mechanism included the transfer of exosomal ANXA6 from resistant cancer cells to sensitive cancer cells. Isobaric peptide labeling–liquid chromatography–tandem mass spectrometry and western blotting revealed that ANXA6 was upregulated in resistant cancer cells and their derived exosomes. Sensitive cancer cells exhibited resistance with increased viability and colony formation and decreased apoptosis when ANXA6 was stably overexpressed. On the contrary, knockdown ANXA6 restored the sensitivity of cells to gemcitabine. Co-immunoprecipitation expression and GST pulldown assay demonstrated that exosomal ANXA6 and EGFR could interact with each other and exosomal ANXA6 was associated with the suppression of EGFR ubiquitination and downregulation. While adding lapatinib reversed gemcitabine resistance induced by exosomal ANXA6. Moreover, ANXA6 and EGFR protein expression was correlated in TNBC tissues, and exosomal ANXA6 levels at baseline were lower in patients with highly sensitive TNBC than those with resistant TNBC when treated with first-line gemcitabine-based chemotherapy. In conclusion, resistant cancer cell-derived exosomes induced gemcitabine resistance via exosomal ANXA6, which was associated with the inhibition of EGFR ubiquitination and degradation. Exosomal ANXA6 levels in the serum of patients with TNBC might be predictive of the response to gemcitabine-based chemotherapy.Subject terms: Breast cancer, Predictive markers     

Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL® platform

Background

The c D NA-mediated A nnealing, extension, S election and L igation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel^v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.

Methods

Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance t -statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI).

Results

Technical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values < 0.05 and log2 -fold changes > |0.5| in expression between HER2 + and HER2 - tumors: topoisomerase II alpha (TOP2A), cyclin a2 (CCNA2), v-fos fbj murine osteosarcoma viral oncogene homolog (FOS), wingless-type mmtv integration site family, member 5a (WNT5A), growth factor receptor-bound protein 7 (GRB7), cell division cycle 2 (CDC2), and baculoviral iap repeat-containing protein 5 (BIRC5). The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around v-myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein p53 (TP53), and estrogen receptor α (ESR1). Network analysis with a two-step extension also showed that the eight discriminating genes common to the 1.5K and 24K panels are functionally linked together through MYC, TP53, and ESR1.

Conclusions

The relative RNA abundance obtained from two highly differing density gene panels are correlated with eight common genes differentiating HER2 + and HER2 - breast tumors. Network analyses demonstrated biological consistency between the 1.5K and 24K gene panels.     

Kinetics of gene expression and bone remodelling in the clinical phase of collagen-induced arthritis

IntroductionPathological bone changes differ considerably between inflammatory arthritic diseases and most studies have focused on bone erosion. Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis, which, in addition to bone erosion, demonstrates bone formation at the time of clinical manifestations. The objective of this study was to use this model to characterise the histological and molecular changes in bone remodelling, and relate these to the clinical disease development.MethodsA histological and gene expression profiling time-course study on bone remodelling in CIA was linked to onset of clinical symptoms. Global gene expression was studied with a gene chip array system.ResultsThe main histopathological changes in bone structure and inflammation occurred during the first two weeks following the onset of clinical symptoms in the joint. Hereafter, the inflammation declined and remodelling of formed bone dominated.Global gene expression profiling showed simultaneous upregulation of genes related to bone changes and inflammation in week 0 to 2 after onset of clinical disease. Furthermore, we observed time-dependent expression of genes involved in early and late osteoblast differentiation and function, which mirrored the histopathological bone changes. The differentially expressed genes belong to the bone morphogenetic pathway (BMP) and, in addition, include the osteoblast markers integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap1), and secreted phosphoprotein 1 (Spp1). Pregnancy-associated protein A (Pappa) and periostin (Postn), differentially expressed in the early disease phase, are proposed to participate in bone formation, and we suggest that they play a role in early bone formation in the CIA model. Comparison to human genome-wide association studies (GWAS) revealed differential expression of several genes associated with human arthritis.ConclusionsIn the CIA model, bone formation in the joint starts shortly after onset of clinical symptoms, which results in bony fusion within one to two weeks. This makes it a candidate model for investigating the relationship between inflammation and bone formation in inflammatory arthritis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0531-7) contains supplementary material, which is available to authorized users.     

Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans

X-linked sex-ratio distorters that disrupt spermatogenesis can cause a deficiency in functional Y-bearing sperm and a female-biased sex ratio. Y-linked modifiers that restore a normal sex ratio might be abundant and favored when a X-linked distorter is present. Here we investigated natural variation of Y-linked suppressors of sex-ratio in the Winters systems and the ability of these chromosomes to modulate gene expression in Drosophila simulans. Seventy-eight Y chromosomes of worldwide origin were assayed for their resistance to the X-linked sex-ratio distorter gene Dox. Y chromosome diversity caused males to sire ∼63% to ∼98% female progeny. Genome-wide gene expression analysis revealed hundreds of genes differentially expressed between isogenic males with sensitive (high sex ratio) and resistant (low sex ratio) Y chromosomes from the same population. Although the expression of about 75% of all testis-specific genes remained unchanged across Y chromosomes, a subset of post-meiotic genes was upregulated by resistant Y chromosomes. Conversely, a set of accessory gland-specific genes and mitochondrial genes were downregulated in males with resistant Y chromosomes. The D. simulans Y chromosome also modulated gene expression in XXY females in which the Y-linked protein-coding genes are not transcribed. The data suggest that the Y chromosome might exert its regulatory functions through epigenetic mechanisms that do not require the expression of protein-coding genes. The gene network that modulates sex ratio distortion by the Y chromosome is poorly understood, other than that it might include interactions with mitochondria and enriched for genes expressed in post-meiotic stages of spermatogenesis.     

OLFM4 depletion sensitizes gallbladder cancer cells to cisplatin through the ARL6IP1/caspase-3 axis

BackgroundGallbladder cancer (GBC) is a highly lethal malignancy that carries an extremely poor prognosis due to its chemoresistant nature. Cisplatin (CDDP) is a first-line chemotherapeutic for GBC; however, patients experienced no benefit when treated with CDDP alone. The underlying mechanisms of CDDP resistance in GBC remain largely unknown.MethodsAgilent mRNA microarray analysis was performed between paired GBC and paracarcinoma to explore differentially expressed genes that might underlie drug resistance. Gene Set Enrichment Analysis (GSEA) was employed to identify key genes mediating CDDP resistance in GBC, and immunohistochemistry was performed to validate protein expression and test correlations with clinicopathological features. In vitro and in vivo functional assays were performed to investigate the proteins’ roles in CDDP resistance.ResultsOlfactomedin 4 (OLFM4) was differentially expressed between GBC and paracarcinoma and had the highest rank metric score in the GSEA. OLFM4 expression was increasingly upregulated from chronic cholecystitis to GBC in clinical tissue samples, and OLFM4 depletion decreased GBC cell proliferation and invasion. Interestingly, downregulation of OLFM4 reduced ARL6IP1 (antiapoptotic factor) expression and sensitized GBC cells to CDDP both in vitro and in vivo. The evidence indicated that CDDP could significantly increase Bax and Bad expression and activate caspase-3 cascade in OLFM4-depleted GBC cells through ARL6IP1. Clinically, lower OLFM4 expression was associated with good prognosis of GBC patients.ConclusionsOur results suggest that OLFM4 is an essential gene that contributes to GBC chemoresistance and could serve as a prognostic biomarker for GBC. Importantly, OLFM4 could be a potential chemotherapeutic target.     

Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.     

Comparative RNA-Seq analysis reveals insights in Salmonella disease resistance of chicken; and database development as resource for gene expression in poultry

Salmonella, one of the major infectious diseases in poultry, causes considerable economic losses in terms of mortality and morbidity, especially in countries that lack effective vaccination programs. Besides being resistant to diseases, indigenous chicken breeds are also a potential source of animal protein in developing countries. For understanding the disease resistance, an indigenous chicken line Kashmir faverolla, and commercial broiler were selected. RNA-seq was performed after challenging the chicken with Salmonella Typhimurium. Comparative differential expression results showed that following infection, a total of 3153 genes and 1787 genes were differentially expressed in the liver and spleen, respectively. The genes that were differentially expressed included interleukins, cytokines, NOS2, Avβ-defensins, toll-like receptors, and other immune-related gene families. Most of the genes and signaling pathways involved in the innate and adaptive immune responses against bacterial infection were significantly enriched in the Kashmir faverolla. Pathway analysis revealed that most of the enriched pathways were MAPK signaling pathway, NOD-like receptor signaling pathway, TLR signaling pathway, PPAR signaling pathway, endocytosis, etc. Surprisingly some immune-related genes like TLRs were upregulated in the susceptible chicken breed. On postmortem examination, the resistant birds showed small lesions in the liver compared to large necrotic lesions in susceptible birds. The pathological manifestations and RNA sequencing results suggest a balancing link between resistance and infection tolerance in Kashmir faverolla. Here we also developed an online Poultry Infection Database (https://skuastk.org/pif/index.html), the first publicly available gene expression resource for disease resistance in chickens. The available database not only shows the data for gene expression in chicken tissues but also provides quick search, visualization and download capacity.