Background: Cathepsin L (Ctsl) is a cysteine protease mainly located within the endosomal/lysosomal cell compartment. High expression of Ctsl indicates poor prognosis in human breast cancer. However, the cell type-specific Ctsl functions responsible for this association remain elusive. Methods: Because constitutive Ctsl^−/− mice develop a complex phenotype, we developed a conditional model allowing for cell type-specific inactivation of Ctsl in mammary epithelium or myeloid cells in the transgenic mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) breast cancer model. Results: Ctsl ablation in mammary epithelial cells resulted in delayed initiation and end-stage of cancers. The latter displayed large dead cell areas. Inducible in vitro deletion of Ctsl in MMTV-PyMT-derived breast cancer cells revealed expansion of the acidic cell compartment, alteration of intracellular amino acid levels, and impaired mTOR signaling. In consequence, Ctsl-deficient cells exhibited slow growth rates and high apoptosis susceptibility. In contrast to Ctsl-deficient mammary epithelium, selective knockout of Ctsl in myeloid cells had no effects on primary tumors, but promoted lung metastasis formation. Conclusions: Our cell type-specific in vivo analysis provides strong evidence for a cancer cell-intrinsic, tumor-promoting role of Ctsl in primary breast cancer, whereas metastasis is negatively regulated by Ctsl expressed by bone marrow-derived cells.

XD14 is a 4-acyl pyrrole derivative, which was discovered by a high-throughput virtual screening experiment. XD14 inhibits bromodomain and extra-terminal domain (BET) proteins (BRD2, BRD3, BRD4 and BRDT) and consequently suppresses cell proliferation. In this study, metabolic profiling reveals the molecular effects in the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) treated by XD14. A three-day time series experiment with two concentrations of XD14 was performed. Gas chromatography-mass spectrometry (GC-MS) was applied for untargeted profiling of treated and non-treated MCF-7 cells. The gained data sets were evaluated by several statistical methods: analysis of variance (ANOVA), clustering analysis, principle component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). Cell proliferation was strongly inhibited by treatment with 50 µM XD14. Samples could be discriminated by time and XD14 concentration using PLS-DA. From the 117 identified metabolites, 67 were significantly altered after XD14 treatment. These metabolites include amino acids, fatty acids, Krebs cycle and glycolysis intermediates, as well as compounds of purine and pyrimidine metabolism. This massive intervention in energy metabolism and the lack of available nucleotides could explain the decreased proliferation rate of the cancer cells.

Forced expression of four transcription factors are sufficient to reprogram mouse embryonic fibroblasts (MEFs) directly to induced renal tubular epithelial cells (iRECs). These cells have been characterized as tubule cells by transcriptomic, morphological and functional studies. Recently, we analyzed iRECs by untargeted metabolomics and thereby proved their cellular identity. Hence, changes by application of a common nephrotoxic agent confirmed many alterations occurring in vivo [in submission]. In this study, we investigated the impact of glucose on MEFs and iRECs by conducting an untargeted gas chromatography/mass spectrometry based profiling with high and low glucose concentrations. Whereas accumulating in MEFs used for reprogramming, glucose was efficiently metabolized by glycolysis and citric acid cycle in iRECs but also an increase in the polyol pathway was observed. The activation of this pathway and a consequent generation of reactive oxygen species is a common phenomenon in diabetic complications such as diabetic retinopathy, neuropathy and nephropathy (DN). Thus, iRECs transpired to be an excellent in vitro model for tubule damage, an aspect of DN being overshadowed by the glomerular focus. The possibility to generate iRECs also from human fibroblasts holds great potential in patient specific testing for exogenous challenges in general.