Magdalena Schlotter

BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1

Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma.

Molecular-cytogenetic comparison of mucosa-associated marginal zone B-cell lymphoma and large B-cell lymphoma arising in the gastro-intestinal tract

Extranodal B‐cell lymphoma of mucosa‐associated lymphoid tissue (MALT) type may represent a model of lymphoma progression, because a small cell component frequently occurs in the large cell variants. We studied 52 extranodal B‐cell lymphomas: 18 extranodal marginal zone B‐cell lymphomas of MALT type (MZBL,MT), 7 MZBL,MT of the gastro‐intestinal tract with a diffuse large B‐cell component (giMZBLplusLBCL), and 27 diffuse large B‐cell lymphomas of the gastro‐intestinal tract without small cell component (giLBCL). Analytical techniques were comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The translocation t(11;18) was found as the sole aberration in two MZBL,MT only. In contrast to this, t(11;18)‐negative MZBL,MT were characterized by frequent gains on chromosome 3 and DNA amplifications on 2p13–p15. Furthermore, we found a clonal lymphoma progression from the small to the large cell component with accumulation of gains and losses of chromosomal material in the large cell component in giMZBLplusLBCL. Aberrations overlapping with MZBL,MT and giMZBLplusLBCL included losses on chromosome 13, amplifications of the REL proto‐oncogene, or gains on chromosome 12. In addition, the large cell component revealed gains on 8q24, including amplifications of the MYC proto‐oncogene, and losses on 2q. The giLBCL had frequent gains on chromosomes 12 and 9, as well as on 11q, and losses on 6q. We conclude that, based on the distinctive and partly overlapping patterns of genetic aberrations, MALT lymphomas can be divided into different genetic subgroups.

Translocation t(X;11)(q13;q23) in B-cell chronic lymphocytic leukemia disrupts two novel genes†‡

Deletion of chromosome region 11q22–q23 defines a subgroup of patients with B‐cell chronic lymphocytic leukemia (B‐CLL) characterized by poor survival. Although the tumor‐suppressor gene ATM in the consensus deletion region was found to be biallelically inactivated in about one third of B‐CLL cases, in the majority of those who have this deletion, inactivation of the remaining ATM allele was not observed. To identify a second disease‐associated gene, we investigated two B‐CLL cases with translocation breakpoints in the critical 11q23 deletion region. In one case, a t(X;11)(q13;q23) was cloned and two novel genes were isolated. The breakpoint on 11q23 affected the ARHGAP20 gene, which encodes a protein predicted to be involved in the regulation of Rho family GTPases. The breakpoint on Xq13 occurred in BRWD3, which codes for a putative novel transcription factor. The rearrangement of ARHGAP20 and BRWD3 did not result in fusion transcripts, but it disrupted both genes. Mutation analysis of 28 B‐CLL samples with monoallelic deletions and two B‐CLL samples with 11q23 translocations detected no deleterious mutation in the remaining copy of ARHGAP20. Quantitative expression analysis in 22 B‐CLLs revealed significant up‐regulation of ARHGAP20 in CLL B cells, whereas BRWD3 was slightly down‐regulated. Thus, deregulation of ARHGAP20 by altered gene expression or by gene disruption (but not point mutation) might be a general molecular mechanism of B‐CLL leukemogenesis.

Reprogramming of the ERRα and ERα Target Gene Landscape Triggers Tamoxifen Resistance in Breast Cancer

Endocrine treatment regimens for breast cancer that target the estrogen receptor-α (ERα) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-α (ERRα) for ERα. To examine this hypothesis, we analyzed ERRα and ERα in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERα, ERRα, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERα and ERRα target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRα in tamoxifen- and fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRα sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n = 1041), increased expression of ERRα was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERα-positive cases. In addition, increased ERRα expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERα and ERRα cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRα as a candidate drug target to treat endocrine-resistant breast cancer.

A gene signature distinguishing CD133hi from CD133- colorectal cancer cells: essential role for EGR1 and downstream factors

Aims: In colorectal cancer (CRC), CD133 expression is an independent prognostic marker associated with adverse clinical outcome. The CD133 epitope AC133 allowed isolating stem cells from normal and cancerous tissues, although its use in colon was questioned. We aimed to identify differences between AC133hi and AC133− cells. Methods: We analysed the gene expression profiles of EpCAM+/CEA+/AC133hi and EpCAM+/CEA+/AC133− cells from primary CRC and liver metastasis tissues (n = 5). Immunohistochemistry confirmed these results in a validation set. Results: We identified 68 genes differentially expressed between both populations, including genes of notorious importance in CRC pathogenesis, and several candidates not previously shown to play a major role in CRC. Notably, EGR1 belonged to the most highly expressed genes in AC133hi cells. In the validation set, the presence of EGR1 and CD133 correlated (r = 0.625). Since EGR1 regulates Wnt through up-regulation of TCF4, which induces stem cell marker LGR5, the potential association between LGR5, EGR1 and CD133 was investigated. The presence of LGR5 correlated with the presence of EGR1 and CD133. Strong signals for LGR5 were detected throughout tumour invasion fronts. Conclusions: The study suggests a connection between CD133 and EGR1 and emphasises the importance of the EGR1/TCF4/CD133/LGR5 network in CRC.

The branched-chain amino acid transaminase 1 sustains growth of antiestrogen-resistant and ERα-negative breast cancer

Antiestrogen-resistant and triple-negative breast tumors pose a serious clinical challenge because of limited treatment options. We assessed global gene expression changes in antiestrogen-sensitive compared with antiestrogen-resistant (two tamoxifen resistant and two fulvestrant resistant) MCF-7 breast cancer cell lines. The branched-chain amino acid transaminase 1 (BCAT1), which catalyzes the first step in the breakdown of branched-chain amino acids, was among the most upregulated transcripts in antiestrogen-resistant cells. Elevated BCAT1 expression was confirmed in relapsed tamoxifen-resistant breast tumor specimens. High intratumoral BCAT1 levels were associated with a reduced relapse-free survival in adjuvant tamoxifen-treated patients and overall survival in unselected patients. On a tissue microarray (n=1421), BCAT1 expression was detectable in 58% of unselected primary breast carcinomas and linked to a higher Ki-67 proliferation index, as well as histological grade. Interestingly, BCAT1 was predominantly expressed in estrogen receptor-α-negative/human epidermal growth factor receptor-2-positive (ERα-negative/HER-2-positive) and triple-negative breast cancers in independent patient cohorts. The inverse relationship between BCAT1 and ERα was corroborated in various breast cancer cell lines and pharmacological long-term depletion of ERα induced BCAT1 expression in vitro. Mechanistically, BCAT1 indirectly controlled expression of the cell cycle inhibitor p27Kip1 thereby affecting pRB. Correspondingly, phenotypic analyses using a lentiviral-mediated BCAT1 short hairpin RNA knockdown revealed that BCAT1 sustains proliferation in addition to migration and invasion and that its overexpression enhanced the capacity of antiestrogen-sensitive cells to grow in the presence of antiestrogens. Importantly, silencing of BCAT1 in an orthotopic triple-negative xenograft model resulted in a massive reduction of tumor volume in vivo, supporting our findings that BCAT1 is necessary for the growth of hormone-independent breast tumors.

Low-dose Actinomycin-D treatment re-establishes the tumoursuppressive function of P53 in RELA -positive ependymoma

Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA-fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity. In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2–0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis (PUMA) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation.

Branched‐chain ketoacids secreted by glioblastoma cells via MCT1 modulate macrophage phenotype

Elevated amino acid catabolism is common to many cancers. Here, we show that glioblastoma are excreting large amounts of branched‐chain ketoacids (BCKAs), metabolites of branched‐chain amino acid (BCAA) catabolism. We show that efflux of BCKAs, as well as pyruvate, is mediated by the monocarboxylate transporter 1 (MCT1) in glioblastoma. MCT1 locates in close proximity to BCKA‐generating branched‐chain amino acid transaminase 1, suggesting possible functional interaction of the proteins. Using in vitro models, we demonstrate that tumor‐excreted BCKAs can be taken up and re‐aminated to BCAAs by tumor‐associated macrophages. Furthermore, exposure to BCKAs reduced the phagocytic activity of macrophages. This study provides further evidence for the eminent role of BCAA catabolism in glioblastoma by demonstrating that tumor‐excreted BCKAs might have a direct role in tumor immune suppression. Our data further suggest that the anti‐proliferative effects of MCT1 knockdown observed by others might be related to the blocked excretion of BCKAs.