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Background: Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making.
Methods: In this contribution we will show that the combination of the three label-free non-linear imaging modalities CARS (coherent anti-Stokes Raman-scattering), TPEF (two-photon excited autofluorescence) and SHG (second harmonic generation) yields information that can be translated into computational hematoxylin and eosin (HE) images by multivariate statistics. Thereby, a computational HE stain is generated resulting in pseudo-HE overview images that allow for identification of suspicious regions. The latter are analyzed further by Raman-spectroscopy retrieving the tissue’s molecular fingerprint.
Results: The results suggest that the combination of non-linear multimodal imaging and Raman-spectroscopy possesses the potential as a precise and fast tool in routine histopathology.
Conclusions: As the key advantage, both optical methods are non-invasive enabling for further pathological investigations of the same tissue section, e.g. a direct comparison with the current pathological gold-standard.
Gallbladder cancer (GBC) is a highly malignant tumor characterized by a poor response to chemotherapy and radiotherapy. We evaluated the in vitro and in vivo antitumor efficacy of mTOR inhibitors, rapamycin and WYE-354. In vitro assays showed WYE-354 significantly reduced cell viability, migration and invasion and phospho-P70S6K expression in GBC cells. Mice harboring subcutaneous gallbladder tumors, treated with WYE-354 or rapamycin, exhibited a significant reduction in tumor mass. A short-term treatment with a higher dose of WYE-354 decreased the tumor size by 68.6% and 52.4%, in mice harboring G-415 or TGBC-2TKB tumors, respectively, compared to the control group. By contrast, treatment with a prolonged-low-dose regime of rapamycin almost abrogated tumor growth, exhibiting 92.7% and 97.1% reduction in tumor size, respectively, compared to control mice. These results were accompanied by a greater decrease in the phosphorylation status of P70S6K and a lower cell proliferation Ki67 index, compared to WYE-354 treated mice, suggesting a more effective mTOR pathway inhibition. These findings provide a proof of concept for the use of rapamycin or WYE-354 as potentially good candidates to be studied in clinical trials in GBC patients.
MiR144/451 expression is repressed by RUNX1 during megakaryopoiesis and disturbed by RUNX1/ETO
(2016)
Abstract: A network of lineage-specific transcription factors and microRNAs tightly regulates differentiation of hematopoietic stem cells along the distinct lineages. Deregulation of this regulatory network contributes to impaired lineage fidelity and leukemogenesis. We found that the hematopoietic master regulator RUNX1 controls the expression of certain microRNAs, of importance during erythroid/megakaryocytic differentiation. In particular, we show that the erythorid miR144/451 cluster is epigenetically repressed by RUNX1 during megakaryopoiesis. Furthermore, the leukemogenic RUNX1/ETO fusion protein transcriptionally represses the miR144/451 pre-microRNA. Thus RUNX1/ETO contributes to increased expression of miR451 target genes and interferes with normal gene expression during differentiation. Furthermore, we observed that inhibition of RUNX1/ETO in Kasumi1 cells and in RUNX1/ETO positive primary acute myeloid leukemia patient samples leads to up-regulation of miR144/451. RUNX1 thus emerges as a key regulator of a microRNA network, driving differentiation at the megakaryocytic/erythroid branching point. The network is disturbed by the leukemogenic RUNX1/ETO fusion product.
Author Summary: The regulatory network between transcription factors, epigenetic cofactors and microRNAs is decisive for normal hematopoiesis. The transcription factor RUNX1 is important for the establishment of a megakaryocytic gene expression program and the concomitant repression of erythroid genes during megakaryocytic differentiation. Gene regulation by RUNX1 is frequently disturbed by mutations and chromosomal translocations, such as the t(8;21) translocation, which gives rise to the leukemogenic RUNX1/ETO fusion protein. We found that RUNX1 regulates microRNAs, which are of importance at the megakaryocytic/erythroid branching point. Specifically, RUNX1 down-regulates expression of the microRNA cluster miR144/451 during megakaryocytic differentiation by changing the epigenetic histone modification pattern at the locus. We could show that miR451, one of the micorRNAs of the miR144/451 cluster, supports erythroid differentiation. We found that expression of miR451 is repressed by the RUNX1/ETO fusion protein, resulting in up regulation of miR451 target genes. Our data support the notion that RUNX1 suppresses the erythroid gene expression program including the erythroid microRNA miR451 and that the RUNX1/ETO fusion protein interferes with normal gene regulation by RUNX1.
The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4, which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast, at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia.
Immunohistochemical assessment of phosphorylated mTORC1-pathway proteins in human brain tumors
(2015)
Background: Current pathological diagnostics include the analysis of (epi-)genetic alterations as well as oncogenic pathways. Deregulated mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated in a variety of cancers including malignant gliomas and is considered a promising target in cancer treatment. Monitoring of mTORC1 activity before and during inhibitor therapy is essential. The aim of our study is to provide a recommendation and report on pitfalls in the use of phospho-specific antibodies against mTORC1-targets phospho-RPS6 (Ser235/236; Ser240/244) and phospho-4EBP1 (Thr37/46) in formalin fixed, paraffin embedded material.
Methods and findings: Primary, established cell lines and brain tumor tissue from routine diagnostics were assessed by immunocyto-, immunohistochemistry, immunofluorescent stainings and immunoblotting. For validation of results, immunoblotting experiments were performed. mTORC-pathway activation was pharmacologically inhibited by torin2 and rapamycin. Torin2 treatment led to a strong reduction of signal intensity and frequency of all tested antibodies. In contrast phospho-4EBP1 did not show considerable reduction in staining intensity after rapamycin treatment, while immunocytochemistry with both phospho-RPS6-specific antibodies showed a reduced signal compared to controls. Staining intensity of both phospho-RPS6-specific antibodies did not show considerable decrease in stability in a timeline from 0–230 minutes without tissue fixation, however we observed a strong decrease of staining intensity in phospho-4EBP1 after 30 minutes. Detection of phospho-signals was strongly dependent on tissue size and fixation gradient. mTORC1-signaling was significantly induced in glioblastomas although not restricted to cancer cells but also detectable in non-neoplastic cells.
Conclusion: Here we provide a recommendation for phospho-specific immunohistochemistry for patient-orientated therapy decisions and monitoring treatment response.
Background: The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation.
Methods: Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation.
Results: The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity.
Conclusion: Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy.
Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo.
IKKα promotes intestinal tumorigenesis by limiting recruitment of M1-like polarized myeloid cells
(2014)
The recruitment of immune cells into solid tumors is an essential prerequisite of tumor development. Depending on the prevailing polarization profile of these infiltrating leucocytes, tumorigenesis is either promoted or blocked. Here, we identify IκB kinase α (IKKα) as a central regulator of a tumoricidal microenvironment during intestinal carcinogenesis. Mice deficient in IKKα kinase activity are largely protected from intestinal tumor development that is dependent on the enhanced recruitment of interferon γ (IFNγ)-expressing M1-like myeloid cells. In IKKα mutant mice, M1-like polarization is not controlled in a cell-autonomous manner but, rather, depends on the interplay of both IKKα mutant tumor epithelia and immune cells. Because therapies aiming at the tumor microenvironment rather than directly at the mutated cancer cell may circumvent resistance development, we suggest IKKα as a promising target for colorectal cancer (CRC) therapy.
Myelodysplastic syndromes (MDSs) represent clonal disorders mainly of the elderly that are characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. The pathogenesis of MDS is thought to evolve from accumulation and selection of specific genetic or epigenetic events. Emerging evidence indicates that MDS is not solely a hematopoietic disease but rather affects the entire bone marrow microenvironment, including bone metabolism. Many of these cells, in particular mesenchymal stem and progenitor cells (MSPCs) and osteoblasts, express a number of adhesion molecules and secreted factors that regulate blood regeneration throughout life by contributing to hematopoietic stem and progenitor cell (HSPC) maintenance, self-renewal and differentiation. Several endocrine factors, such as erythropoietin, parathyroid hormone and estrogens, as well as deranged iron metabolism modulate these processes. Thus, interactions between MSPC and HSPC contribute to the pathogenesis of MDS and associated pathologies. A detailed understanding of these mechanisms may help to define novel targets for diagnosis and possibly therapy. In this review, we will discuss the scientific rationale of "osteohematology" as an emerging research field in MDS and outline clinical implications.
The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long non-coding RNA (lncRNA) that has been discovered as a marker for lung cancer metastasis. It is highly abundant, its expression is strongly regulated in many tumor entities including lung adenocarcinoma and hepatocellular carcinoma as well as physiological processes, and it is associated with many RNA binding proteins and highly conserved throughout evolution. The nuclear transcript MALAT-1 has been functionally associated with gene regulation and alternative splicing and its regulation has been shown to impact proliferation, apoptosis, migration and invasion.
Here, we have developed a human and a mouse knockout system to study the loss-of-function phenotypes of this important ncRNA. In human tumor cells, MALAT1 expression was abrogated using Zinc Finger Nucleases. Unexpectedly, the quantitative loss of MALAT1 did neither affect proliferation nor cell cycle progression nor nuclear architecture in human lung or liver cancer cells. Moreover, genetic loss of Malat1 in a knockout mouse model did not give rise to any obvious phenotype or histological abnormalities in Malat1-null compared with wild-type animals. Thus, loss of the abundant nuclear long ncRNA MALAT1 is compatible with cell viability and normal development.