Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair.

The melanocytic lineage, which is prominently exposed to ultraviolet radiation (UVR) and radiation-independent oxidative damage, requires specific DNA-damage response mechanisms to maintain genomic and transcriptional homeostasis. The coordinate lineage-specific regulation of intricately intertwined DNA repair and transcription is incompletely understood. Here we demonstrate that the Microphthalmia-associated transcription factor (MITF) directly controls general transcription and UVR-induced nucleotide excision repair by transactivation of GTF2H1 as a core element of TFIIH. Thus, MITF ensures the rapid resumption of transcription after completion of strand repair and maintains transcriptional output, which is indispensable for survival of the melanocytic lineage including melanoma in vitro and in vivo. Moreover, MITF controls c-MYC implicated in general transcription by transactivation of far upstream binding protein 2 (FUBP2/KSHRP), which induces c-MYC pulse regulation through TFIIH, and experimental depletion of MITF results in consecutive loss of CDK7 in the TFIIH-CAK subcomplex. Targeted for proteasomal degradation, CDK7 is dependent on transactivation by MITF or c-MYC to maintain a steady state. The dependence of TFIIH-CAK on sequence-specific MITF and c-MYC constitutes a previously unrecognized mechanism feeding into super-enhancer-driven or other oncogenic transcriptional circuitries, which supports the concept of a transcription-directed therapeutic intervention in melanoma.

Development and Characterization of a Spontaneously Metastatic Patient-Derived Xenograft Model of Human Prostate Cancer.

Here we describe the establishment and characterization of an AR+, PSMA+, ERG+, PTEN-/-, CHD1+/- patient-derived xenograft (PDX) model termed 'C5', which has been developed from a 60 years old patient suffering from castration-resistant prostate cancer (CRPC). The patient underwent radical prostatectomy, showed early tumor marker PSA recurrence and, one year after surgery, abiraterone resistance. Subcutaneous C5 tumors can be serially transplanted between mice and grow within ~90 days to 1.5-2 cm³ tumors in SCID Balb/c mice (take rate 100%), NOD-scid IL2Rgnull (NSG) mice (100%) and C57BL/6 pfp-/-/rag2-/- mice (66%). In contrast, no tumor growth is observed in female mice. C5 tumors can be cryopreserved and show the same growth characteristics in vivo afterwards. C5 tumor cells do not grow stably in vitro, neither under two- nor three-dimensional cell culture conditions. Upon serial transplantation, some C5 tumors spontaneously disseminated to distant sites with an observable trend towards higher metastatic cell loads in scid compared to NSG mice. Lung metastases could be verified by histology by means of anti-PSMA immunohistochemistry, exclusively demonstrating single disseminated tumor cells (DTCs) and micro-metastases. Upon surgical resection of the primary tumors, such pulmonary foci rarely grew out to multi-cellular metastatic colonies despite doubled overall survival span. In the brain and bone marrow, the metastatic cell load present at surgery even disappeared during the post-surgical period. We provide shallow whole genome sequencing and whole exome sequencing data of C5 tumors demonstrating the copy number aberration/ mutation status of this PCa model and proving genomic stability over several passages. Moreover, we analyzed genomic and transcriptomic alterations during metastatic progression achieved by serial transplantation. This study describes a novel PCa PDX model that enables future research on several aspects of metastatic PCa, particularly for the AR+ , ERG+ , PTEN-/- PCa subtype.

CEACAM1 promotes melanoma metastasis and is involved in the regulation of the EMT associated gene network in melanoma cells.

We investigated the functional role of CEACAM1 in a spontaneous metastasis xenograft model of human melanoma in scid mice using BRAF wildtype MeWo cells with and without RNAi mediated knockdown of CEACAM1. Tumors from the xenograft model were subjected to whole genome expression analysis and metastasis was quantified histologically. Results and identified markers were verified using tissue samples of over 100 melanoma patients. Knockdown of CEACAM1 prolonged the animals' survival by significantly reducing subcutaneous growth of MeWo tumors and spontaneous lung metastasis. Microarray analysis revealed a strong influence of CEACAM1 knockdown on the network of EMT associated genes in the xenograft tumors (e.g. downregulation of BRAF, FOSL1, NRAS and TWIST). IGFBP7 and Latexin (highest up- and downregulated expression in microarray analysis) were found to be associated with longer and shorter survival, respectively, of melanoma patients. High FOSL1 and altered TWIST1 expression were found to be correlated with shortened survival in the cohort of melanoma patients. After a stepwise selection procedure combining above markers, multivariate analysis revealed IGFBP7, Latexin and altered TWIST to be prognostic markers for death. CEACAM1 could be a target for melanoma therapy as an alternative to (or in combination with) immune checkpoint and BRAF inhibitors.

Human prostate cancer in a clinically relevant xenograft mouse model: identification of ß(1,6)-branched oligosaccharides as a marker of tumor progression.

PURPOSE: To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients. EXPERIMENTAL DESIGN: Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xenografted subcutaneously into immunodeficient pfp(-/-)/rag2(-/-) mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples. RESULTS: PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of ß1,6-N-acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [ß(1,6)-branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival. CONCLUSION: We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of ß(1,6)-branched oligosaccharides for prostate cancer progression.

Brachyury expression predicts poor prognosis at early stages of colorectal cancer.

Although survival rates of colon cancer patients diagnosed at an early stage (T1-2N0M0; Dukes A) vary considerably according to the studies cited, several studies indicate development of distant metastases already occurring in a considerable percentage of these patients leading to the death of the patients. This particular high risk group cannot be identified properly as no marker exists to identify these patients. As the Wnt/Win pathway plays a crucial role in metastasis formation in colorectal carcinoma, we analysed whether the transcription factor brachyury critically involved in this pathway may predict metastasis formation in these patients. The expression of brachyury-homologous (T) was immunohistochemically analysed in 748 patients and the data were correlated with classical and newer prognostic markers in colorectal cancer. Early stages colorectal cancer patients (T1-2N0M0, Dukes A) showed a significantly decreased survival when brachyury was expressed in the tumour tissue while no correlation was observed in later tumour stages. Hence a subset of colorectal cancers exists in which the ability to metastasise is already present at early stages of tumour growth and this high risk group can now be detected by immunohistochemistry.

Limited tumor growth (HT29) in vivo under RO205-2349 is due to increased apoptosis and reduced cell volume but not to decreased proliferation rate.

The peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that plays a regulatory role in cell differentiation and proliferation. PPARgamma was first detected in adipocytes, however, it has been shown that this receptor is also expressed in normal as well as tumor cells including malignant colonic epithelial cells. In this study, the effect of the PPARgamma agonist RO205-2349, a recently developed thiazolidinedione, on tumor growth was evaluated. For this purpose, human colon cancer cells (HT29) were grown in severe combined immunodeficient mice. Under daily RO205-2349 treatment (50 mg/kg/day) a significantly reduced tumor weight became evident after 3 weeks. In the control (n = 10) and treatment (n = 10) groups the mean tumor weights were 0.45 and 0.16 g, respectively. The mean percentages of apoptotic cells were 0.8 and 2.7% in the control and treatment groups, respectively, and the cell diameter measured on average 11.4 and 9.4 microm. In contrast, cell proliferation and differentiation, which are considered to be influenced by the PPARgamma, remained unaffected as could be seen by Ki-67 and carcinoembryonic antigen immunoreactivity indicating that increased rate of apoptosis and cell shrinkage are responsible for the differences in tumor growth. Hence, in this human/mouse xenograft model, mechanisms other than the classical activation of PPARgamma are likely reasons causing limited tumor growth.