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Zusammenfassung  Die Krebsstammzelle ist elementar von ihrem Homingareal (Mikroenvironment) geprägt und abhängig. Die Kenntnis der Hüllfaszien erleichtert eine operative Entfernung des Homingareals. Zu ihm gehören neben dem Fettgewebe u.a. die regionären Lymphknoten. Primärkrebse und deren Lokalrezidive verharren in ihren Homingarealen. Nur ihre gelungene operative Entfernung verspricht lang anhaltende Symptomfreiheit. Im Tumor, auch im Lokalrezidiv, bilden sich Lymphgefäße, je maligner, desto mehr. Auch die Anzahl befallener Lymphknoten ist vom Malignitätsgrad abhängig und damit ist die Prognose festgelegt. Eine folgende Untersuchung weist nach, dass das Grading im mikromolekularen Bereich nach den Erbgesetzen abläuft. Adjuvante Maßnahmen z. B. bei Tumorstreuung (Fernmetastasen) provozieren eine Zellauslese. [ABSTRACT FROM AUTHOR]

Abstract Sarcomatous malignancies only rarely develop regional lymph node metastases: about 2.7% of our evaluated cases. In this paper we provide evidence supporting a new hypothesis that two entirely separate lymph vascular systems exist in humans. One system (LGS I) exists in close proximity to the epithelium and drains into regional lymph nodes. Only sarcomas that originate in the epithelium or its immediate proximity are able to form regional lymph node metastases. The vast majority of sarcomatous malignancies (97.4% of cases) do not give rise to lymph node metastases, since they originate in proximity to a second, more deeply localized lymph node system (LGS II) in the mesenchymally derived tissues of the body. This second system has no connection to regional lymph nodes. Supporting evidence is provided by experience in the operative treatment of extremity lymphedema, PET-CT examinations, radionuclear lymphography, and scientific investigations using antibodies specifically directed at the elements of the lymph vascular system. [ABSTRACT FROM AUTHOR]

Photorespiration, caused by oxygenation of the enzyme Rubisco, is considered a wasteful process, because it reduces photosynthetic carbon gain, but it also supplies amino acids and is involved in amelioration of stress. Here, we show that a sudden increase in photorespiratory activity not only reduced carbon acquisition and production of sugars and starch, but also affected diurnal dynamics of amino acids not obviously involved in the process. Flux calculations based on diurnal metabolite profiles suggest that export of proline from leaves increases, while aspartate family members accumulate. An immense increase is observed for turnover in the cyclic reaction of glutamine synthetase/glutamine-oxoglutarate aminotransferase (GS/GOGAT), probably because of increased production of ammonium in photorespiration. The hpr1-1 mutant, defective in peroxisomal hydroxypyruvate reductase, shows substantial alterations in flux, leading to a shift from the oxoglutarate to the aspartate family of amino acids. This is coupled to a massive export of asparagine, which may serve in exchange for serine between shoot and root.

Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings’ CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient’s CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd.

Echinococcus multilocularis is endemic in Germany. However, alveolar echinococcosis is a rare disease. Most commonly the parasite affects the liver, behaving like a malignant tumour. Bones are affected in less than 2% of cases. We report a case of vertebral osteomyelitis accompanied by recurrent cutaneous fistula formation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

MET amplifications (METamp) occur in 5% of NSCLC and represent in most case mechanisms of resistance to ALK and/or EGFR-targeted therapies. METamp detection can be performed using different techniques, although Fluorescence In-Situ Hybridization (FISH) remains the gold-standard, especially in the context of subclonality. To date current evaluation algorithms of MET amplifications are time consuming. Aim of the study was to identify a faster, equally reliable diagnostic algorithm for the detection of METamp, which is currently classified in negativity and low/intermediate/high-level amplification. N=497 NSCLC cases with available MET-FISH data had been selected. The results based on the first evaluated 20 cells had been re-calculated and compared with the definitive results based on 60 cells. For n=464 (93.4%) identical results had been obtained when counting 20 cells instead of 60 cells. Thirty-three cases (5.6%) showed a discrepancy, leading to an incorrect upgrade to a higher diagnostic category (n=25) and to an incorrect downgrade (n=8). We propose a simplified, yet equally reliable MET FISH-algorithm: after accurate screening of the whole tumor slide, twenty tumor cells have to be evaluated and results calculated: If the result is negative, or if all criteria of high-level METamp are fulfilled, the case can be signed out as such. All other cases should be considered as equivocal and additional 40 cells have to be counted. Given that, reliable results can be obtained by counting 20 cells only and an "equivocal" category for cases that need further investigation have been clearly defined., Competing Interests: Conflict of Interest RB provided lectures and was part of Advisory Boards for AbbVie, Amgen, AstraZeneca, Bayer, BMS, Boehringer-Ingelheim, Illumina, Lilly, Merck-Serono, MSD, Novartis, Qiagen, Pfizer, Roche, Targos MP Inc. RB is Co-Founder and Scientific Advisor for Targos Mol. Pathology Inc. RB is Testifying Advisor for MSD in GBA-Assessment for Pembrolizumab. RB has received funding from the Deutsche Krebshilfe for the Network Genomic Medicine. SMB has received speaker honoraria and personal fees from Pfizer, Novartis, Roche, Bayer, AstraZeneca, Molecular Health, GSK, MSD and Targos; speaker honoraria and non-financial support from BMS; non-financial support from Janssen. The authors declare no further conflict of interest. RC was supported by the Else Kröner-Fresenius Stiftung (2016-Kolleg.19). AMS, CA and BH were supported by Roche Pharma AG. The authors have no further conflict of interest to disclaim.

Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is caused by germline mutations of mismatch-repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. MLH1-/PMS2-deficient colorectal carcinomas might be HNPCC-associated but also caused by MLH1-promoter methylation in sporadic colon carcinoma. This study analyzed semiquantitatively whether the MLH1 staining pattern might be indicative of sporadic or HNPCC-associated colorectal cancer. Using a semiquantitative score ranging from 0 (negative) to 12 (maximum immunopositivity) we analyzed MLH1 expression patterns in 130 MLH1-/PMS2-deficient colorectal cancers. The collective consisted of 70 HNPCC-associated colorectal cancers and 60 sporadic colon cancers. In tumor cells of 70 HNPCC-associated colorectal cancers, 64 cases (91.43%) showed no MLH1 staining, 5 cases weak (7.14%) and 1 case (1.43%) stronger staining intensity. In contrast, in tumor cells of 60 sporadic colorectal cancers 45 cases (75.0%) showed no MLH1 staining, 10 cases weak (16.67%) and 5 cases (8.33%) stronger staining intensity to a varying extent. In immunopositive cases, MLH1 showed a characteristic dot-like nuclear staining pattern in the tumor cells. We compared cases with absent to weak MLH1-staining (immunoscores 0 to 2) to cases with elevated immunoscores (3 to 12) detecting a statistically significant difference between HNPCC-associated and sporadic colon cancers (p value = 0.0031, Fisher's exact test). Taken together, enhanced tumoral MLH1 expression in MLH1-/PMS2-deficient colorectal carcinomas seems to be indicative of sporadic origin. In contrast, HNPCC-associated colorectal cancer showed absent or very weak MLH1 immunopositivity. Therefore, this semiquantitative and easy to exert MLH1 immunoscore might help to identify sporadic MLH1-/PMS2-deficient colorectal cancer cases prior to time-consuming methylation analysis.

Introduction: Colorectal cancers (CRCs) deficient in the DNA mismatch repair protein MutL homolog 1 (MLH1) display distinct clinicopathological features and require a different therapeutic approach compared to CRCs with MLH1 proficiency. However, the molecular basis of this fundamental difference remains elusive. Here, we report that MLH1-deficient CRCs exhibit reduced levels of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1), and that tumor progression and metastasis of CRCs correlate with SPTAN1 levels., Methods and Results: To investigate the link between MLH1 and SPTAN1 in cancer progression, a cohort of 189 patients with CRC was analyzed by immunohistochemistry. Compared with the surrounding normal mucosa, SPTAN1 expression was reduced in MLH1-deficient CRCs, whereas MLH1-proficient CRCs showed a significant upregulation of SPTAN1. Overall, we identified a strong correlation between MLH1 status and SPTAN1 expression. When comparing TNM classification and SPTAN1 levels, we found higher SPTAN1 levels in stage I CRCs, while stages II to IV showed a gradual reduction of SPTAN1 expression. In addition, SPTAN1 expression was lower in metastatic compared with non-metastatic CRCs. Knockdown of SPTAN1 in CRC cell lines demonstrated decreased cell viability, impaired cellular mobility and reduced cell-cell contact formation, indicating that SPTAN1 plays an important role in cell growth and cell attachment. The observed weakened cell-cell contact of SPTAN1 knockdown cells might indicate that tumor cells expressing low levels of SPTAN1 detach from their primary tumor and metastasize more easily., Conclusion: Taken together, we demonstrate that MLH1 deficiency, low SPTAN1 expression, and tumor progression and metastasis are in close relation. We conclude that SPTAN1 is a candidate molecule explaining the tumor progression and metastasis of MLH1-deficient CRCs. The detailed analysis of SPTAN1 is now mandatory to substantiate its relevance and its potential value as a candidate protein for targeted therapy, and as a predictive marker of cancer aggressiveness., Competing Interests: The authors have declared that no competing interests exist.

In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background: PD-L1 immunohistochemistry is predictive for molecular inhibitors of PD-1/PD-L1 immune checkpoint. Therefore, this study evaluated the PD-L1 expression in patients with Hereditary Non-Polyposis Colorectal Cancer (HNPCC)., Methods: Immunohistochemical expression of PD-L1 in carcinoma cells, stromal macrophages and lymphocytes of 40 HNPCC-patients with colorectal cancer was scored semi-quantitatively., Results: Focal (2 cases) to extensive (2 cases) PD-L1-immunopositivity of carcinoma cells was detected in 4 out of 40 cases (10.0%). Stromal macrophages were immunopositive in 28 out of 40 cases (70.0%). Lymphocytes showed PD-L1 expression in 3 out of 40 cases (7.5%). Simultaneous immunopositivity of stromal macrophages and tumor cells was detected in two MLH1/PMS2-deficient and two MSH2/MSH6-deficient cases., Conclusion: A subset of HNPCC-associated colorectal cancers in this study clearly showed PD-L1 expression of tumor epithelia and immune cells, therefore, the detection of PD-L1 status is useful., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

In ∼30% of families affected by colorectal adenomatous polyposis, no germline mutations have been identified in the previously implicated genes APC, MUTYH, POLE, POLD1, and NTHL1, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, we performed exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis. We identified two unrelated individuals with differing compound-heterozygous loss-of-function (LoF) germline mutations in the mismatch-repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1G>A, c.2760delC, and c.3001-2A>C) was indicated at the RNA and protein levels. Analysis of the diseased individuals' tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST), and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the LoF effect and causal relevance of the mutations. The pedigrees, genotypes, and frequency of MSH3 mutations in the general population are consistent with an autosomal-recessive mode of inheritance. Both index persons have an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing constitutional mismatch-repair deficiency. Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study, we identified biallelic germline MSH3 mutations in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Objective: Microsatellite instability (MSI) is detected in approximately 15% of all colorectal cancers (CRC) and virtually in all cases with Lynch syndrome. The MSI phenotype is caused by dysfunctional mismatch repair (MMR) and leads to accumulation of DNA replication errors. Sporadic MSI CRC often harbours BRAF(V600E); however, no consistent data exist regarding targeted treatment approaches in BRAF(wt) MSI CRC., Design: Mutations and quantitative MSI were analysed by deep sequencing in 196 formalin fixed paraffin embedded (FFPE) specimens comprising Lynch and Lynch-like CRCs from the German Hereditary Nonpolyposis Colorectal Cancer registry. Functional relevance of recurrent ERBB2/HER2 mutations was investigated in CRC cell lines using reversible and irreversible HER-targeting inhibitors, EGFR-directed antibody cetuximab, HER2-directed antibody trastuzumab and siRNA-mediated ERBB2/HER2 knockdown., Results: Quantification of nucleotide loss in non-coding mononucleotide repeats distinguished microsatellite status with very high accuracy (area under curve=0.9998) and demonstrated progressive losses with deeper invasion of MMR-deficient colorectal neoplasms (p=0.008). Characterisation of BRAF(wt) MSI CRC revealed hot-spot mutations in well-known oncogenic drivers, including KRAS (38.7%), PIK3CA (36.5%), and ERBB2 (15.0%). L755S and V842I substitutions in ERBB2 were highly recurrent. Functional analyses in ERBB2-mutated MSI CRC cell lines revealed a differential response to HER-targeting compounds and superiority of irreversible pan-HER inhibitors., Conclusions: We developed a high-throughput deep sequencing approach for concomitant MSI and mutational analyses in FFPE specimens. We provided novel insights into clinically relevant alterations in MSI CRC and a rationale for targeting ERBB2/HER2 mutations in Lynch and Lynch-like CRC., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Pilomatricoma is a tumour derived from hair matrix cells, which shows progressive keratin expression. Tumorigenesis is frequently associated with activating mutations in β-catenin gene inducing nuclear expression of β-catenin protein. The present study analysed the role of transforming growth factor-β1 (TGF-β1) and four-and-a-half LIM domain protein 2 (FHL2) in pilomatricoma in synopsis with their expression patterns in human anagen hair. Human anagen hair showed TGF-β1 and nuclear FHL2 expression in the outer root sheath layer separated from nuclear β-catenin staining, which was observed in cells of matrix and inner root sheath layers. Correspondingly, 41 out of 50 pilomatricomas showed co-labelling of TGF-β1 and nuclear FHL2 in tumour cells, which mostly lacked nuclear β-catenin expression. Tumoural proliferation (ki67) was associated with nuclear β-catenin staining but not with expression of nuclear FHL2. In early pilomatricomas, TGF-β1 expression was observed in few peripheral tumour cells showing absent or faint nuclear FHL2 co-staining. TGF-β1 expression extended in growing tumours going along with strong nuclear FHL2 co-labelling as well as progressive keratin 14 and keratin 1 expression. In vitro, cultured human keratinocytes showed weak to marked autocrine TGF-β1 expression; in case of enhanced TGF-β1 expression associated with keratin 10 staining. TGF-β1-treatment of cultured human keratinocytes induced nuclear and cytoplasmatic FHL2 staining as well as keratin 14 staining. Accordingly, siRNA-mediated FHL2 knockdown of TGF-β1-stimulated keratinocytes reduced keratin 14 staining. In conclusion, tumoural TGF-β1 secretion seems to induce nuclear translocation of co-factor FHL2 mediating progressive keratin expression in pilomatricoma.

Objectives: This study examines the expression and the role of four-and-a-half LIM domains protein 2 (FHL2) and transforming growth factor β1 (TGF-β1) in human malignant melanoma. It is determined whether both proteins influence melanoma survival time., Methods: We analyzed the immunohistochemical staining intensities of FHL2 and TGF-β1 in normal skin and in 50 malignant melanomas with different mutation status (BRAF-V600E, NRAS codon 61 mutation, and wild type). Survival data were available for 45 cases., Results: In melanocytes of nonneoplastic human skin, FHL2 expression was absent. In contrast, 38 (76%) of 50 melanomas showed strong cytoplasmic and partly nuclear FHL2 expression. At the invasion front, cytoplasmic TGF-β1 staining was observed in 32 (64%) of 50 melanomas, and a correlation of FHL2 and TGF-β1 staining intensities was detectable. In follow-up analyses, enhanced FHL2 and TGF-β1 staining intensities in the tumor invasion front were associated with poor survival., Conclusions: Enhanced FHL2 and TGF-β1 expression is correlated with poor survival in human malignant melanoma. Protumorigenic effects of autocrine TGF-β1 secretion might be exerted by induction of FHL2 expression in melanoma cells. Since melanomas treated with targeted therapies often do not show sufficient response rates, inhibition of FHL2 and/or TGF-β1 might be a promising therapeutic approach., (Copyright© by the American Society for Clinical Pathology.)

Background: Four and a half LIM domain protein-2 (FHL2) is part of the focal adhesion structures modulating cell motility. FHL2 may translocate into the nucleus serving as a transcriptional cofactor binding several transcription factors. Overexpression of FHL2 has been linked to cancer progression in various neoplasias. The aim of the present study was to determine, whether FHL2's function as nuclear cofactor plays a prognostic role in invading tumor cells of sporadic and HNPCC-associated colorectal cancer (CRC)., Design: Immunohistochemical staining intensity of nuclear FHL2 was quantified by Remmele score analysing 47 sporadic and 42 HNPCC-associated colorectal cancers. Analysis was restricted to carcinoma cells of the tumoral invasion front., Results: Confocal microscopy detected nuclear expression of FHL2 in colon cancer cells and absence of nuclear FHL2 signal in normal colon enterocytes. In colon cancer, nuclear FHL2 expression was predominantly observed in low-differentiated, often mucinous tumor areas. 42.55% of sporadic and 54.76% of HNPCC-associated CRC showed enhanced (Remmele score 6-12) nuclear FHL2 expression in the carcinoma cells of the tumoral advancing edge. Enhanced nuclear FHL2 expression was significantly linked to lymphatic metastasis in sporadic CRC (p=0.0197) and almost reached significance in HNPCC-associated CRC (p=0.0545). In contrast, nuclear FHL2 expression was neither associated with hematogenic metastasis in sporadic (p=0.7087) nor in HNPCC-associated colorectal cancer (p=0.3007)., Conclusions: We recently demonstrated that enhanced nuclear FHL2 expression in tumor stroma of sporadic colon cancer is associated with lymphatic metastasis. The results of the present study indicate a synergistic effect of nuclear cofactor FHL2 in tumor cells as well as in peritumoral stroma cells promoting lymphatic metastasis in sporadic CRC. As HNPCC-associated tumors did not show a significant association between tumoral nuclear FHL2 expression and lymphatic metastasis we speculate, that the intensive lymphocytic immune response in HNPCC precludes a direct contact of tumor cells and stromal cells resulting in reduced lymphatic spread., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Synovial sarcoma is a high-grade soft tissue malignancy characterized by a specific reciprocal translocation t(X;18), which leads to the fusion of the SS18 (SYT) gene to one of three SSX genes (SSX1, SSX2 or SSX4). The resulting chimeric SS18-SSX protein is suggested to act as an oncogenic transcriptional regulator. Despite multimodal therapeutic approaches, metastatic disease is often lethal and the development of novel targeted therapeutic strategies is required. Several expression-profiling studies identified distinct gene expression signatures, implying a consistent role of Wnt/β-catenin signaling in synovial sarcoma tumorigenesis. Here we investigate the functional and therapeutic relevance of Wnt/β-catenin pathway activation in vitro and in vivo. Immunohistochemical analyses of nuclear β-catenin and Wnt downstream targets revealed activation of canonical Wnt signaling in a significant subset of 30 primary synovial sarcoma specimens. Functional aspects of Wnt signaling including dependence of Tcf/β-catenin complex activity on the SS18-SSX fusion proteins were analyzed. Efficient SS18-SSX-dependent activation of the Tcf/β-catenin transcriptional complex was confirmed by TOPflash reporter luciferase assays and immunoblotting. In five human synovial sarcoma cell lines, inhibition of the Tcf/β-catenin protein-protein interaction significantly blocked the canonical Wnt/β-catenin signaling cascade, accompanied by the effective downregulation of Wnt targets (AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin) and the specific suppression of cell viability associated with the induction of apoptosis. In SYO-1 synovial sarcoma xenografts, administration of small molecule Tcf/β-catenin complex inhibitors significantly reduced tumor growth, associated with diminished AXIN2 protein levels. In summary, SS18-SSX-induced Wnt/β-catenin signaling appears to be of crucial biological importance in synovial sarcoma tumorigenesis and progression, representing a potential molecular target for the development of novel therapeutic strategies.

Exposure to environmental cues such as cold or nutritional imbalance requires white adipose tissue (WAT) to adapt its metabolism to ensure survival. Metabolic plasticity is prominently exemplified by the enhancement of mitochondrial biogenesis in WAT in response to cold exposure or β3-adrenergic stimulation. Here we show that these stimuli increase the levels of lysine-specific demethylase 1 (LSD1) in WAT of mice and that elevated LSD1 levels induce mitochondrial activity. Genome-wide binding and transcriptome analyses demonstrate that LSD1 directly stimulates the expression of genes involved in oxidative phosphorylation (OXPHOS) in cooperation with nuclear respiratory factor 1 (Nrf1). In transgenic (Tg) mice, increased levels of LSD1 promote in a cell-autonomous manner the formation of islets of metabolically active brown-like adipocytes in WAT. Notably, Tg mice show limited weight gain when fed a high-fat diet. Taken together, our data establish LSD1 as a key regulator of OXPHOS and metabolic adaptation in WAT.

Introduction: Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated., Methods: Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively., Results: MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability., Conclusions: These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.

Synovial sarcoma is a soft-tissue malignancy characterized by a reciprocal t(X;18) translocation encoding a chimeric transcriptional modifier. Several receptor tyrosine kinases have been found activated in synovial sarcoma; however, no convincing therapeutic concept has emerged from these findings. On the basis of the results of phosphokinase screening arrays, we here investigate the functional and therapeutic relevance of the SRC kinase in synovial sarcoma. Immunohistochemistry of phosphorylated SRC and its regulators CSK and PTP1B (PTPN1) was conducted in 30 synovial sarcomas. Functional aspects of SRC, including dependence of SRC activation on the SS18/SSX fusion proteins, were analyzed in vitro. Eventually, synovial sarcoma xenografts were treated with the SRC inhibitor dasatinib in vivo. Activated phospho (p)-(Tyr416)-SRC was detected in the majority of tumors; dysregulation of CSK or PTP1B was excluded as the reason for the activation of the kinase. Expression of the SS18/SSX fusion proteins in T-REx-293 cells was associated with increased p-(Tyr416)-SRC levels, linked with an induction of the insulin-like growth factor pathway. Treatment of synovial sarcoma cells with dasatinib led to apoptosis and inhibition of cellular proliferation, associated with reduced phosphorylation of FAK (PTK2), STAT3, IGF-IR, and AKT. Concurrent exposure of cells to dasatinib and chemotherapeutic agents resulted in additive effects. Cellular migration and invasion were dependent on signals transmitted by SRC involving regulation of the Rho GTPases Rac and RhoA. Treatment of nude mice with SYO-1 xenografts with dasatinib significantly inhibited tumor growth in vivo. In summary, SRC is of crucial biologic importance and represents a promising therapeutic target in synovial sarcoma., (©2013 AACR.)

Four and a half LIM domain protein-2 (FHL2) is a component of the focal adhesion structures and has been suggested to have an important role in cancer progression. This study analyses the role of FHL2 in peritumoural fibroblasts of sporadic and hereditary non-polyposis colorectal cancer (HNPCC). Tissue specimens of 48 sporadic and 49 hereditary colon cancers, respectively, were stained immunohistochemically for FHL2, transforming growth factor (TGF)-β1 ligand and α-SMA. Myofibroblasts at the tumour invasion front co-expressed α-SMA and FHL2. Sporadic colon cancer but not HNPCC cases showed a correlation between TGF-β1 expression of the invading tumour cells and FHL2 staining of peritumoural myofibroblasts. Overexpression of FHL2 in peritumoural myofibroblasts correlated to lymphatic metastasis in sporadic colon cancer but not in HNPCC. In cultured mouse fibroblasts, TGF-β1 treatment induced myofibroblast differentiation, stimulated FHL2 protein expression and elevated number of migratory cells in transwell motility assays, suggesting that FHL2 is regulated downstream of TGF-β. Physical contact of colon cancer cells and myofibroblasts via FHL2-positive focal adhesions was detected in human colon carcinoma tissue and in co-culture assays using sporadic as well as HNPCC-derived tumour cell lines. Our data provide strong evidence for an important role of FHL2 in the progression of colon cancers. Tumour-secreted TGF-β1 stimulates FHL2 protein expression in peritumoural fibroblasts, probably facilitating the invasion of tumour glands into the surrounding tissue by enhanced myofibroblast migration and tight connection of fibroblasts to tumour cells via focal adhesions. These findings are absent in HNPCC-associated colon cancers in vivo and may contribute to a less invasive and more protruding tumour margin of microsatellite instable carcinomas.

Synovial sarcomas account for 5-10% of all malignant soft tissue tumors. They have been shown to express different membranous growth factor receptors, many of them signaling via intracellular kinase cascades. In our study, the functional role of PI3K/AKT signals in synovial sarcoma is analyzed with regard to tumor biology and therapeutic applicability. Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT, its targets p-(Ser9)-GSK-3β and p-(Ser2448)-mTOR and the cell cycle regulators Cyclin D1 and p27(KIP1) were performed in 36 synovial sarcomas. The PIK3CA gene was screened for mutations. In vitro, four synovial sarcoma cell lines were treated with the PI3K inhibitor LY294002. Phosphorylation of AKT, GSK-3β and mTOR was assessed, and cellular proliferation and apoptosis were analyzed to functionally characterize the effects of PI3K inhibition. Finally, coincubations of LY294002 with cytotoxic drugs were performed. Most tumors showed significant expression levels of p-AKT, p-GSK-3β and p-mTOR, indicating activation of the PI3K/AKT signaling cascade in synovial sarcomas; Cyclin D1 and p27(KIP1) were differentially expressed. Mutations in the PIK3CA gene could be excluded. In vitro, PI3K inhibition diminished synovial sarcoma cell growth accompanied by reduced phosphorylation of AKT, GSK-3β and mTOR. Mechanistically, PI3K pathway inhibition lead to enhanced apoptosis and decreased cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) levels. Simultaneous treatment of synovial sarcoma cell lines with LY294002 and cytotoxic drugs resulted in additive effects. In summary, PI3K signaling plays an essential role in growth control of synovial sarcomas and might be successfully targeted in multimodal therapeutic strategies., (Copyright © 2010 UICC.)

Background: The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed., Methodology/principal Findings: Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh(-/-) mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh(-/-) mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh(+/+) and Ctsh(-/-) mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh(-/-) mice., Conclusions/significance: We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.

Preoperative neoadjuvant chemotherapy (NAC) can significantly reduce tumour burden in patients with primarily unresectable chemosensitive tumours, allowing a more complete cytoreduction during debulking surgery and facilitating evaluation of tumour chemosensitivity, identification of appropriate treatment options and improvement of intervention protocols. In this study, we investigate, using immunohistochemistry, the impact of platinum/taxane-based NAC (NAC) on tumour-infiltrating lymphocytes (TILs) in advanced epithelial ovarian cancer (EOC) and their relationship with clinical outcome. All patients had clinical response, as shown by ascites volume and CA125 levels compared to pre-treatment findings. NAC intervention significantly increased CD4(+), CD8(+) and granzyme B(+) infiltration while Foxp3(+) accumulation remained unaffected. TILs were prognostically neutral for both progression-free survival (PFS) and overall survival (OS) before NAC. In contrast, after NAC, elevated granzyme B(+) infiltration displayed a tendency for improved PFS (log-rank 0.064). Further, low Foxp3(+) cell density was associated with longer PFS, as compared with strong Foxp3(+) infiltration (median 20.94 vs. 11.24 months; log-rank 0.0001) and with improved OS (median 30.75 vs. 16.04 months, respectively; log-rank 0.056), demonstrating clear prognostic significance for PFS. In addition, high granzyme B(+)/Foxp3(+) ratio post-NAC strongly correlated with improved PFS compared to low granzyme B(+)/Foxp3(+) cell ratio (median 17.88 vs. 11.24 months, respectively), and showed to be a favourable prognostic factor for PFS (log-rank 0.014). Our findings indicate that NAC elicited an immunologic profile in which low immunosuppressive Foxp3(+) infiltration and elevated numbers of activated granzyme B(+) cells were significantly associated with EOC-specific PFS, suggesting a contribution of immunologic effects to improved clinical outcome.

Objective: To evaluate changes in Ki-67 expression during neoadjuvant chemotherapy (NACT) in advanced ovarian cancer., Materials and Methods: Patients with International Federation of Gynecology and Obstetrics stage IIIC or IV and large-volume ascites were treated with NACT within a phase 2 trial. The expression of Ki-67 was evaluated by immunohistochemistry on paraffin-embedded tissue samples and classified by percentage of stained cells. Survival curves were plotted using the Kaplan-Meier method., Results: Comparison of 40 individual paired results from pretreatment and posttreatment samples revealed a median difference of -0.11 in the Ki-67 index (95% confidence interval, -0.20 to -0.01; P = 0.005, signed rank test). Univariate analysis identified a high Ki-67 index as well as an increasing Ki-67 index after NACT as significant prognostic markers for progression-free survival (P = 0.004 and P = 0.001; log-rank test). Six of 12 patients with an increased Ki-67 index after NACT developed recurrence within 6 months after therapy., Conclusions: Changes of the Ki-67 index during NACT are associated with progression-free survival. If confirmed in prospective trials, an increasing Ki-67 index during preoperative treatment may serve as an indicator for resistance to chemotherapy.