Clonal heterogeneity in ER+ breast cancer reveals the proteasome and PKC as potential therapeutic targets.

Intratumoral heterogeneity impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By isolating single clones from the resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and potentially shared sensitivities for repression of protein kinase C. Our in vitro findings are consistent with tumor-heterogeneity that is observed in breast cancer patients thus highlighting the need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.

Abrogating GPT2 in triple-negative breast cancer inhibits tumor growth and promotes autophagy.

Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.

Time-Resolved Profiling Reveals ATF3 as a Novel Mediator of Endocrine Resistance in Breast Cancer.

Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor α account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, depending on the degree of severity of the disease at diagnosis, 10 to 40% of these tumors eventually relapse due to resistance development. Even though recent novel approaches as the combination with CDK4/6 inhibitors increased the overall survival of relapsing patients, this remains relatively short and there is a urgent need to find alternative targetable pathways. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle, and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model, and elevated expression of ATF3 was verified in patient datasets, adding clinical relevance to our findings. This study proposes ATF3 as a novel mediator of endocrine resistance development in breast cancer and elucidates its role in the regulation of downstream pathways activities.

miRNA-1246 induces pro-inflammatory responses in mesenchymal stem/stromal cells by regulating PKA and PP2A.

The tumor microenvironment (TME) has an impact on breast cancer progression by creating a pro-inflammatory milieu within the tumor. However, little is known about the roles of miRNAs in cells of the TME during this process. We identified six putative oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells (MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent of TNFα stimulations and mediated by direct targeting of the tumor-suppressors PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3 phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7, T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancer-related inflammation and breast cancer progression.

The highly expressed 5'isomiR of hsa-miR-140-3p contributes to the tumor-suppressive effects of miR-140 by reducing breast cancer proliferation and migration.

BACKGROUND: miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, the so-called 5'isomiRs exhibit a shifted 5' end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. RESULTS: Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5'isomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5'isomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. miRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5'isomiR-140-3p were highly expressed in patients' tumors compared to normal breast tissue. In the current work, we present the functional characterization of 5'isomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A, MDA-MB-468 and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa-miR-140-3p, overexpression of the 5'isomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5'isomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5'ismoiR-140-3p overexpression was found to cause a decrease in cell migration in the three cell lines. We identified three novel direct target genes of the 5'isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5'isomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. CONCLUSIONS: In summary, this work presents evidence that there is functional synergy between the canonical hsa-miR-140-3p and the newly identified 5'isomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration.

First synthesis of dimethyl-1H-isochromeno[3,4-b]carbazoles.

The first synthesis of isochromene fused carbazols, (4aS, 13bR)-2,5,5-trimethyl-3,4,4a,5,8,13b-hexahydroisochromeno[3,4-b]carbazole (2) and its epi-isomer 3 by condensation of citral and 2-hydroxycarbazole using Ti(OEt)4 and MeAlC12 as catalysts is described.

1-O-Substituted derivatives of murrayafoline A and their antifungal properties.

The synthesis of some 1-oxygenated derivatives of murrayafoline A (1) and their antifungal properties is reported. Three derivatives, 1-hydroxy-3-methyl-9H-carbazole (2), 1-(3-methylbut-2-enyloxy)-3-methyl-9H-carbazole (3) and 1-(2,3,4,6,-tetra-O-acetyl-alpha-D-O-glucopyranosyl)-3-methyl-9H-carbazole (4), of murrayafoline A were synthesized. Compounds 1 and 2 exhibit strong fungicidal activity against Cladosporium cucumerinum at the dose of 12.5 microg.

The anti-proliferative potency of celecoxib is not a class effect of coxibs.

Celecoxib, a COX-2 (cyclooxygenase-2)-selective inhibitor (coxib), is the only NSAID (non-steroidal anti-inflammatory drug) that has been approved for adjuvant treatment of patients with familial adenomatous polyposis. To investigate if the anti-proliferative effect of celecoxib extends to other coxibs, we compared the anti-proliferative potency of all coxibs currently available (celecoxib, rofecoxib, etoricoxib, valdecoxib, lumiracoxib). Additionally, we used methylcelecoxib (DMC), a close structural analogue of celecoxib lacking COX-2-inhibitory activity. Due to the fact that COX-2 inhibition is the main characteristic of these substances (with exception of methylcelecoxib), we conducted all experiments in COX-2-overexpressing (HCA-7) and COX-2-negative (HCT-116) human colon cancer cells, in order to elucidate whether the observed effects after coxib treatment depend on COX-2 inhibition. Cell survival was assessed using the WST proliferation assay. Apoptosis and cell cycle arrest were determined using flow cytometric and Western blot analysis. The in vitro results were confirmed in vivo using the nude mouse model. Among all coxibs tested, only celecoxib and methylcelecoxib decreased cell survival by induction of cell cycle arrest and apoptosis and reduced the growth of tumor xenografts in nude mice. None of the other coxibs (rofecoxib, etoricoxib, valdecoxib, lumiracoxib) produced anti-proliferative effects, indicating the lack of a class effect and of a role for COX-2. Our data emphasize again the outstanding anti-proliferative activity of celecoxib and its close structural analogue methylcelecoxib in colon carcinoma models in vitro and in vivo.

High throughput production of mouse monoclonal antibodies using antigen microarrays.

Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification. Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated monoclonal antibodies against 68 of the targets (85%), within 6 wk of the primary immunization.

HPV18 E6 and E7 genes affect cell cycle, pRB and p53 of cervical tumor cells and represent prominent candidates for intervention by use peptide nucleic acids (PNAs).

Approximately 100% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early viral genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for tumor therapy. We established an application controlling the E6/E7 expression of the HPV type 18, by using viral gene directed peptide nucleic acids (PNAs). One consequence was the complete change in growth to a stagnated behavior of the HPV 18 positive HeLa-S cells. With flow cytometry, we investigated changes in the cell cycle and expression of the pRB (retinoblastoma) and p53 genes acting as antagonists to E6 and E7. We realized that application of PNAs via intracellular cleavable conjugated peptide carriers mediates specific inhibitory effects and we showed that the combined E6/E7-directed PNA-application mediated a clear morphological change from suspension to adherend state and the cells stopped growth. These data could demonstrate a promising approach for development of new 'anti-gene therapeutics' against papillomavirus-induced human cancers.

Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy.

Dunning R3327 AT-1 rat prostate tumor cells were transfected with a double-fusion suicide gene (CDglyTK) that coded for the cytosine deaminase from E. coli and the thymidine kinase (TK) from HSV-1. The resulting cell line AT-1/CDglyTK was incubated with 10 and 20 microg/ml 5-FC or 0.25 microg/ml GCV, or both 5-FC and GCV 96 hours before harvest. The MTS assay detected cell viabilities of 50+/-5 and 25+/-5% after 5-FC treatment, and 50+/-5% after GCV treatment. The dye exclusion and the colony-forming assay confirmed the data of the MTS assay with GCV (47+/-5 and 32+/-5%), but presented different results for the 5-FC incubation. We detected 100+/-1 and 85+/-5% viable cells after 10 microg/ml 5-FC, and 97+/-1 and 85+/-5% after 20 microg/ml 5-FC treatment, respectively. S-phase arrest in both suicide gene systems was noticeable and a significant increase in cell granularity was observed after incubation with GCV or GCV & 5-FC. This study demonstrates that 5-FC and the metabolized 5-FU act not only as genotoxic reagents, but also as RNA-directed agent, because of the recovery of the cells. On the other hand, a significant S-phase block could be observed after 24 hours incubation with GCV. This short time is enough to incorporate the genotoxic GCV metabolites in the nascent DNA to impair the cell cycle.

Intracellular visualization of prostate cancer using magnetic resonance imaging.

The term "molecular imaging" can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. Is a gene expression magnetic resonance imaging (MRI) possible? Therefore, we have developed a novel intravital and intracellular MRI contrast agent composed of a gadolinium complex, an oligonucleotide sequence [peptide nucleic acid (PNA)], and a transmembrane carrier peptide that is composed of a peptide sequence similar to that of the homeodomain of the Antennapedia protein. The goal of our study was to determine whether this contrast agent could be accumulated in tumor cells in vitro (HeLa cells) and in vivo (Dunning R3327 AT1 rat prostate adenocarcinoma) and whether the specificity of the PNA for the up-regulated c-myc mRNA in the cell's cytoplasm would have an effect on contrast agent retention in the tumor cells. Using the c-myc-specific and a c-myc-nonspecific control PNA, an increase in signal intensity in the tumor cells was observed after 10 min in vitro and in vivo (maximum was reached in HeLa cells in vitro in 60 min, in Dunning R3327 AT1 rat prostate adenocarcinoma cells in vivo in 30 min). This increase of signal intensity could be maintained in vitro in HeLa cells for only 4 h and in Dunning R3327 AT1 rat prostate adenocarcinoma cells in vivo at least for 5 h by using the c-myc mRNA-specific PNA as a "retention" agent.

The enhancement of neutron irradiation of HeLa-S cervix carcinoma cells by cell-nucleus-addressed deca-p-boronophenylalanine.

Boron neutron capture therapy (BNCT) is an experimental treatment modality which depends on a sufficient cellular uptake of Boron ((10)B) followed by an exposure to a thermal neutron beam from a nuclear reactor. High energetic particles (4He and 7Li) are created during the neutron capture reaction and produce DNA damages, which lead to cell killing. Regarding BNCT, the short radiation range of He- and Li-particles is decisive for the distribution of (10)B. Until now, BNCT has been lacking for therapeutically effective concentrations of (10)B. Twenty-four hours after the combined use of our 'Bioshuttle'-p-borono-phenylalanine(10)-constructs ('Bioshuttle'-p-BPA(10)) and neutron-irradiation, an obvious reduction of the radiation-resistant HeLa-S cells could be observed. No cells were alive 72 h after the incubation with 'Bioshuttle'-p-BPA(10) followed by neutron irradiation. A post-mitotic cell death could be assumed based on flow cytometrical data.