[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
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Gut 04/2015; DOI:10.1136/gutjnl-2014-309026 · 14.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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).
Pathology - Research and Practice 12/2014; 211(2). DOI:10.1016/j.prp.2014.12.001 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 12/2014; 466(2). DOI:10.1007/s00428-014-1692-5 · 2.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.Oncogene advance online publication, 28 October 2013; doi:10.1038/onc.2013.443.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 alphaII (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.
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.
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.
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.
Molecular Cancer 01/2014; 13(1):11. DOI:10.1186/1476-4598-13-11 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lymph node metastases originating from soft tissue sarcomas are very rare and the reason for this is unclear. While this observation was less important in former times when ultraradical excision and amputation were the norm, modern reconstructive surgical treatment options have to take the possibility of lymphatic metastases into account.We attempted to identify parameters that may be predictive of lymphatic metastases in a cohort of 1,597 patients with soft tissue sarcomas of whom 26 patients (1.6 %) had regional lymph node (RLN) metastases. We studied these RLN metastases with recently described techniques that enabled us to histologically visualize lymphatic vessels.We conclude that sarcomas should not be evaluated from a histogenetic perspective but more on the basis of regional topography of the lymphatic vasculature. As we described previously, two different lymphatic systems should be differentiated: lymphatic vessel system I (LGS I) contains RLN and lymph vessels are mostly superficial; however, there are also vessels near large blood vessels of the extremities. System LGS II is more delicate and its vessels run into the musculature, a metastatic homing area of many sarcomas. Lymph vessels of system LGS II drain directly into veins without intervening lymph nodes. Sarcomas with LGS I drainage will form RLN metastases. In contrast, sarcomas with LGS II drainage will do so only after surgical resection if system LGS I has been opened.
Der Chirurg 04/2013; 84(6). DOI:10.1007/s00104-012-2363-1 · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synovial sarcoma is a rare malignant soft tissue tumor affecting mainly adolescents and young adults. The hallmark of synovial sarcoma is the presence of a reciprocal balanced t(X;18) translocation, leading to the fusion of the SS18 gene to either the SSX1, SSX2 or rarely the SSX4 gene, resulting in a chimeric transcriptional modifier. Therapeutic outcome of synovial sarcomas is primarily determined by the efficiency of surgery as a high tendency for local relapse is documented. Standardized chemo- and radiotherapy are further therapeutic options, however, specific targeted therapies are currently not available. Recently, several expression profiling studies in mesenchymal malignancies revealed gene expression signatures indicating WNT signaling activation in synovial sarcomas. This study was performed to examine the functional relevance of WNT signaling in synovial sarcomas and to evaluate if interference with the WNT signaling pathway might represent an option in the development of novel and highly selective drugs in the treatment of synovial sarcoma. To assess the prevalence of WNT signaling activation in a set of 30 synovial sarcoma tumor samples, nuclear staining of beta-catenin was analyzed immunohistochemically. Nuclear beta-catenin signals were observed in a significant subset of these tumors, indicating activation of the WNT signaling pathway. In order to evaluate whether WNT activation is molecularly dependent on the SS18/SSX fusion proteins, tetracycline-inducible systems overexpressing the SS18/SSX fusion proteins were established in T-Rex293 cells. In luciferase reporter assays employing the TOP-/FOPflash system, expression of SS18/SSX proteins effectively activated TCF/beta-catenin mediated transcriptional activity, which was associated with nuclear recruitment of beta-catenin. Five human synovial sarcoma cell lines were subsequently treated with small molecular inhibitors of WNT signalling. In MTT assays, a significant dose-dependent inhibition of cellular growth was observed, which was accompanied by decreased expression of the WNT downstream targets c-Myc and Cyclin D1. In flow cytometric analyses, the growth effects exerted by the inhibitors were shown to be due to a reduction of cellular proliferation combined with an increase of apoptosis. In summary, our data emphasize the pivotal role of WNT signaling in synovial sarcoma and indicate its functional dependence on the characteristic SS18/SSX translocations. Furthermore, our study demonstrates that targeting the WNT signaling pathway provides a specific, molecularly founded therapeutic strategy in the treatment of synovial sarcoma. Additional functional studies in vitro and in vivo are required to further understand the role of WNT signaling and its therapeutic applicability in synovial sarcomas.
AACR 103rd Annual Meeting of the American Association for Cancer Research; 03/2012
[Show abstract][Hide abstract] ABSTRACT: In 25% aller Kolonkarzinome besteht eine familiäre Häufung. Das hereditäre Non-Polyposis-assoziierte Kolorektalkarzinom (HNPCC)/Lynch-Syndrom ist eine autosomal-dominant vererbte Erkrankung, welche einen Anteil von 2–3% aller kolorektalen Karzinome ausmacht. Da der Übergang von gutartigen Vorstufen zu bösartigen Tumoren und das Tumorwachstum im Vergleich zu sporadisch auftretendem Darmkrebs deutlich beschleunigt abläuft, ist ein gründlicher diagnostischer Nachweis von malignen Neoplasien und deren Vorstufen sowie die standardisierte Überwachung der betroffenen Patienten und ihrer Familien notwendig. Um dies zu leisten, wurde 1999 das deutsche HNPCC Konsortium als ein multizentrisches und interdisziplinäres Netzwerk von 7 Universitäten mit kooperierenden Gastroenterologen, Chirurgen, Humangenetikern, Pathologen und Epidemiologen gegründet.
Die Pathologie liefert die histologische Diagnose und überprüft die Mikrosatelliteninstabilität (MSI) und die Expression der Mismatch-Reparatur-Proteine im Tumorgewebe. Die Diagnose HNPCC gilt als gesichert, wenn bei MSI-Tumoren die Amsterdam-II-Kriterien erfüllt sind oder wenn eine pathogene Keimbahnmutation in den Mismatch-Reparatur-Genen entdeckt wird. Ebenso gilt die Diagnose HNPCC als bestätigt, wenn eine Mutation fehlt, aber Bethesda-Kriterien erfüllt sind und ein Tumor mit MSI oder Verlust der Mismatch-Reparatur-Proteine gefunden wird.
Die Ziele des HNPCC-Konsortiums bestehen in einer verbesserten Überwachung der Indexpatienten und ihrer Familien durch Definition optimaler diagnostischer Intervalle, in der Verbesserung der angewendeten Methoden, in der Entwicklung therapeutischer Ansätze sowie in der Detektion valider prognostischer Marker. Darüber hinaus wird die Integration des HNPCC-Programms in die allgemeine Gesundheitsversorgung angestrebt.
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Cancer 10/2011; 129(7):1564-75. DOI:10.1002/ijc.25829 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Immunology and Immunotherapy 06/2010; 59(6):909-19. DOI:10.1007/s00262-010-0817-1 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate changes in Ki-67 expression during neoadjuvant chemotherapy (NACT) in advanced ovarian cancer.
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.
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.
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.
International Journal of Gynecological Cancer 05/2010; 20(4):555-60. DOI:10.1111/IGC.0b013e3181c104c0 · 1.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of our study was to analyze the effect of taxane-based chemotherapy on tumor angiogenesis in patients with advanced epithelial ovarian cancer.
Within a prospective phase II trial, 32 patients with stage IIIC and IV ovarian cancer were treated with either two or three cycles of neoadjuvant chemotherapy prior to cytoreductive surgery. Carboplatin (AUC5) and docetaxel (75 mg/m2) were administered intravenously in a 3-weekly schedule. Changes in intratumor microvessel density (MVD) were assessed with immunohistochemistry by staining pre- and posttreatment surgical tumor specimens with panendothelial, neovascular and lymphatic vessel markers.
Mean values of MVD defined by CD31, CD34, CD105 and D2-40 antibodies showed 12.3, 21.0, 2.7 and 3.1 vessels per high power field (HPF) before chemotherapy and increased after treatment to 15.3, 21.8, 4.8 and 3.6 per HPF, respectively. These changes were significant for CD31 (p = 0.04) and for CD105 (p = 0.02).
Taxane-based chemotherapy appears to promote tumor vascularization when administered every 3 weeks. A possible explanation is the secondary recovery of MVD in response to immediate cytotoxic and antiangiogenic effects of the chemotherapy. If confirmed prospectively, these findings favor shorter treatment intervals of taxane-based chemotherapy to counteract proangiogenic recovery.
BMC Cancer 04/2010; 10(1):137. DOI:10.1186/1471-2407-10-137 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.
[Show abstract][Hide abstract] ABSTRACT: To study quantitatively the promoter methylation of hMLH1, p16INK, TIMP3 and TPEF genes in patients with colorectal cancer and synchronous polyps, and correlate it with some clinicomorphological features.
DNA was extracted from all studied tumours and the corresponding normal mucosa. Microsatellite instability was analysed using two mononucleotide (BAT 25 and BAT 26) and three dinucleotide markers (D2S123, D5S356, D17S250) and automated DNA sequencing. Quantitative analysis of methylation was performed using DNA bisulfite modification, PCR with biotinylated primers, visualisation by 2% agarose gel electrophoresis and pyrosequencing.
High methylation levels of hMLH1 and p16INK were found in elderly patients (mean age 73.8 +/- 9.5 years and 65.7 +/- 16.6 years, p < 0.03, t-test). Proximal tumours were more often associated with microsatellite instability (p < 0.05, Fisher's test) and higher level of methylation of hMLH1, p16INK and TIMP3 (p < 0.02, Kruskal-Wallis test), while tumours with poor differentiation tended to have higher methylation of the p16INK gene (p < 0.02, Kruskal-Wallis test). Local tumour invasion was correlated with the level of methylation of hMLH1, TIMP3 and the CpG island methylator phenotype (CIMP) status. Tumours with liver metastases showed a lower level of TIMP3 methylation than tumours with no systemic invasion (p < 0.05, Kruskal-Wallis test). We found concordance of methylation in 56% of the cases with colonic cancer and synchronous adenomas; the remaining 44% were discordant.
Tumours with microsatellite instability, high level methylation and CIMP have distinctive clinical and morphological features. The level of hMLH1 and TIMP3 methylation and CIMP status can be correlated with the local tumour invasion. Different mechanisms, even for one and the same patient, can be responsible for the development of more than one third of the synchronous polyps and carcinomas.
[Show abstract][Hide abstract] ABSTRACT: Individuals with hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome) have a high risk for developing colorectal cancer (CRC). We evaluated the efficacy of annual surveillance colonoscopies to detect adenomas and CRCs.
In a prospective, multicenter cohort study, 1126 individuals underwent 3474 colonoscopies. We considered individuals from 3 groups of HNPCC families: those with a pathogenic germline mutation in a mismatch repair gene (MUT group), those without a mutation but with microsatellite instability (MSI group), and those who fulfilled the Amsterdam criteria without microsatellite instability (MSS group).
Compliance to annual intervals was good, with 81% of colonoscopies completed within 15 months. Ninety-nine CRC events were observed in 90 patients. Seventeen CRCs (17%) were detected through symptoms (8 before baseline colonoscopy, 8 at intervals >15 months to the preceding colonoscopy, and 1 interval cancer). Only 2 of 43 CRCs detected by follow-up colonoscopy were regionally advanced. Tumor stages were significantly lower among CRCs detected by follow-up colonoscopies compared with CRCs detected by symptoms (P = .01). Cumulative CRC risk at the age of 60 years was similar in the MUT and MSI groups (23.0% combined; 95% confidence interval [CI], 14.8%-31.2%) but considerably lower in the MSS group (1.8%; 95% CI, 0.0%-5.1%). Adenomas at baseline colonoscopy predicted an earlier occurrence of subsequent adenoma (hazard ratio, 2.6; 95% CI, 1.7-4.0) and CRC (hazard ratio, 3.9; 95% CI, 1.7-8.5), providing information about interindividual heterogeneity of adenomas and kinetics of CRC formation.
Annual colonoscopic surveillance is recommended for individuals with HNPCC. Less intense surveillance might be appropriate for MSS families.
Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 10/2009; 8(2):174-82. DOI:10.1016/j.cgh.2009.10.003 · 7.90 Impact Factor