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Background: We investigated the efficacy and health-related quality of life (HRQoL) in patients receiving either ribociclib plus endocrine therapy (ET) or chemotherapy with/without bevacizumab as first-line treatment of metastatic hormone receptor (HR)-positive, HER2-negative breast cancer (BC)., Patients and Methods: In this randomized, phase III study (RIBBIT), 38 patients diagnosed with metastatic HR-positive, HER2-negative BC with presence of visceral metastases recruited between May 2018 and December 2020 were randomly assigned in a 1:1 ratio to either arm A (ribociclib + ET) or arm B (chemotherapy with/without bevacizumab) at 12 sites in Germany. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall response rate (ORR), overall survival (OS), patient-reported HRQoL, and frequency and type of adverse events. During study conduction, the recruitment rate was persistently and considerably lower than originally expected. Therefore, the recruitment was ended prematurely. The study was initially designed to enroll and randomize 158 patients., Results: Median [95% CI] PFS was 27.3 months [19.1 - NA, parameter not estimable] in arm A and 15.8 months [8.2 - NA] in arm B. Complete responses were achieved only in arm A ( n = 2, 10.5%). The ORR [95% CI] between arm A (57.9% [33.5-79.7]) and arm B (52.6% [28.9-75.6]) was comparable. Median OS [95% CI] was not reached in arm A, while in arm B median OS was 28.4 months [25.0 - NA]. Patients in arm A reported less burden by side-effects. No new safety signals emerged., Conclusion: Treatment of patients with visceral metastatic HR-positive, HER2-negative BC with ribociclib in combination with ET showed a tendency toward a more favorable clinical outcome. Despite small numbers of patients and sites, this head-to-head comparison with chemotherapy supports the use of ribociclib with ET in patients with visceral metastasis at risk of fast disease progression., Competing Interests: Thomas Decker: Honoraria for advisory boards from Novartis, remuneration as principal investigator. Matthias Zaiss: Honoraria for advisory boards from AbbVie, Astra-Zeneca, BMS, Celgene, Esai, Gilead, Hexal, Janssen, Lilly, Novartis, Pfizer, Roche, Vifor. Speaker’s honoraria from MSD, Pfizer, Roche, Takeda, Vifor. Dunja Klein: Employee of iOMEDICO. Antje Hahn: Honoraria from Roche. Volker Hagen: Ownership of company shares from BMS, Johnson & Johnson. Honoraria from Roche, Amgen, Pfizer, Celgene, BMS, FomF, RG Ärztefortbildung, Böhringer Ingelheim, Astra Zeneca. Support for attending meetings and/or travel expenses from Roche, Pfizer, Janssen-Cilag, iOMEDICO, Gilead, Celgene. Paul La Rosée: Honoraria for advisory boards and/or as speaker from Novartis. Rüdiger Liersch: No conflicts of interest. Thomas Wolff: No conflicts of interest. Beate Niemeier: Employee of iOMEDICO. Larissa E Hillebrand: Employee of iOMEDICO. Carolin Lennartz: Employee of iOMEDICO. Marco Chiabudini: Employee of iOMEDICO. Fee Bengsch: Employee of iOMEDICO. Martin Indorf: Employee of iOMEDICO. Norbert Marschner: CSO and shareholder of iOMEDICO., (© 2023 S. Karger AG, Basel.)

Background: Breast cancer is still the most common malignancy in women worldwide. Once metastasized, breast cancer treatment primarily aims at reducing symptom burden, thereby trying to maintain and improve a patient's quality of life (QoL), delaying disease progression, and prolonging survival. Curing the disease is not possible in the palliative setting. To better understand metastatic breast cancer patients, their symptoms, and wishes, which are important for treatment decision making and outcome, patient-reported outcomes (PROs) are of great importance, giving an impression of what really matters to and concerns a patient., Summary: Many advances have been made to implicate PROs in clinical trials, non-interventional studies, registries, and clinical routine care of metastatic breast cancer. For example, large phase III trials like PALOMA-3 (NCT01942135), MONALEESA-7 (NCT02278120), HER2CLIMB (NCT02614794), and KEYNOTE-119 (NCT02555657) trials implemented PROs in their trial design to assess the QoL of their trial patients. Also, non-interventional studies on metastatic breast cancer, e.g., the NABUCCO study (IOM-02240), and prospective non-interventional, multicenter registries, e.g., the tumor registry breast cancer (NCT01351584) or the breast cancer registry platform OPAL (NCT03417115), have implemented PROs to assess QoL during the anticancer treatment periods of the patients., Key Message: Using PROs in metastatic breast cancer can support shared treatment decision making and management of symptoms, eventually leading to an improvement in QoL. Progressively, regulatory authorities take PROs into consideration for the approval of new drugs. Hence, the implication of PROs in cancer treatment, and especially in MBC, is of significant value., (© 2022 S. Karger AG, Basel.)

Previous clinical and experimental evidence strongly supports a breast cancer-promoting function of the lysosomal protease cathepsin B. However, the cathepsin B-dependent molecular pathways are not completely understood. Here, we studied the cathepsin-mediated secretome changes in the context of the MMTV-PyMT breast cancer mouse model. Employing the cell-conditioned media from tumor-macrophage co-cultures, as well as tumor interstitial fluid obtained by a novel strategy from PyMT mice with differential cathepsin B expression, we identified an important proteolytic and lysosomal signature, highlighting the importance of this organelle and these enzymes in the tumor micro-environment. The Cellular Repressor of E1A Stimulated Genes 1 (CREG1), a secreted endolysosomal glycoprotein, displayed reduced abundance upon over-expression of cathepsin B as well as increased abundance upon cathepsin B deletion or inhibition. Moreover, it was cleaved by cathepsin B in vitro. CREG1 reportedly could act as tumor suppressor. We show that treatment of PyMT tumor cells with recombinant CREG1 reduced proliferation, migration, and invasion; whereas, the opposite was observed with reduced CREG1 expression. This was further validated in vivo by orthotopic transplantation. Our study highlights CREG1 as a key player in tumor-stroma interaction and suggests that cathepsin B sustains malignant cell behavior by reducing the levels of the growth suppressor CREG1 in the tumor microenvironment.

Cancer cells within a tumor are heterogeneous and exist in a variety of functionally distinct cell states, which are thought to be hierarchically organized. The cell on top of this hierarchy, the cancer stem cell (CSC) or, alternatively, tumor initiating cell (TIC), is responsible for initiation, maintenance, progression, and relapse of tumors. For the execution of these functions, CSC are equipped with distinct molecular tools. Although proteolytic enzymes in cancers have been extensively studied in general, relatively few studies have addressed proteases in function and fate of CSC/TICs. Here we review protease involvement in cell biological hallmarks of CSC/TICs such as cellular self-renewal, extracellular matrix remodeling and cell motility, resistance to radio- and chemotherapies, as well as evasion of the immune system. In general, CSC/TICs are characterized by a comparatively high expression and activity of proteases. It appears that CSC/TICs install a high degree of pericellular proteolysis depending on metalloproteases such as ADAMs and MMPs but also on secreted serine- and cysteine proteases. Interestingly, it turned out that not all proteases promote the malignant behavior of CSC/TICs. In fact, some proteases, such as ADAM 23, cathepsin K, and granzyme B, have been shown to negatively regulate CSC/TIC functions, thereby exhibiting anti-tumor effects. Finally, we discuss how the enhanced proteolytic signature of CSC/TICs can be used for their therapeutic targeting in order to render this clinically decisive subpopulation of cancer cells harmless., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Tumor-initiating cells (TICs) existing in breast cancer are thought to be involved in initiation, progression, and relapse of tumors. In these processes, the epithelial-to-mesenchymal transition (EMT) and proteases are crucial factors that also dependent on the tumor milieu, including hypoxic nutrient-deprived, as well as normoxic nutrient-rich, environments. Therefore, we investigated EMT and proteases in TICs and their response to different environments by means of a newly generated immortalized TIC (iTIC) line. With the use of primary CD24 + CD90 + CD45 - TICs from the mouse mammary tumor virus-polyoma middle T mouse breast cancer model, iTICs were generated by single cell-initiated sphere and subsequent 2-dimensional monolayer culture. Our data demonstrate the possibility to generate iTICs that are highly tumorigenic in culture and in mouse mammary fat pad. Contrasting environmental conditions provide these cells with a phenotypic and molecular plasticity that has a growth-promoting character in nutrient-rich normoxia and a motile character in nutrient-deprived hypoxia. Expression profiling revealed partial and dynamically changing EMT states, as well as a significantly up-regulated proteolytic signature, including many metalloproteinases, such as matrix metalloproteinase 14 ( Mmp14). Inhibitor treatment of metalloproteinases, as well as short hairpin RNA-mediated knockdown of Mmp14 strongly impacted TIC characteristics, including tumor initiation, cell growth, migration, and invasion, especially in starved environments. We conclude that metalloproteinases empower TICs to adapt to changing environments.-Hillebrand, L. E., Wickberg, S. M., Gomez-Auli, A., Follo, M., Maurer, J., Busch, H., Boerries, M., Reinheckel, T. MMP14 empowers tumor-initiating breast cancer cells under hypoxic nutrient-depleted conditions.

Lysosomal cathepsins are proteolytic enzymes increasingly recognized as prognostic markers and potential therapeutic targets in a variety of diseases. In those conditions, the cathepsins are mostly overexpressed, thereby driving the respective pathogenic processes. Although less known, there are also diseases with a genetic deficiency of cathepsins. In fact, nowadays 6 of the 15 human proteases called 'cathepsins' have been linked to inherited syndromes. However, only three of these syndromes are typical lysosomal storage diseases, while the others are apparently caused by defective cleavage of specific protein substrates. Here, we will provide an introduction on lysosomal cathepsins, followed by a brief description of the clinical symptoms of the various genetic diseases. For each disease, we focus on the known mutations of which many have been only recently identified by modern genome sequencing approaches. We further discuss the effect of the respective mutation on protease structure and activity, the resulting pathogenesis, and possible therapeutic strategies., (© 2016 Federation of European Biochemical Societies.)

Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The cell population, characterized by the marker profile CD24+CD90+CD45-, showed a high tumorigenicity compared to non-CD24+CD90+CD45- cancer cells in colony formation assays, as well as upon orthotopic transplantation into the mammary fat pad of mice. In addition, these orthotopically grown CD24+CD90+CD45- TICs metastasized to the lungs. The transcriptome of TICs freshly isolated from primary tumors by cell sorting was compared with that of sorted non-CD24+CD90+CD45- cancer cells by RNA-seq. In addition to more established TIC signatures, such as epithelial-to-mesenchymal transition or mitogen signaling, an upregulated gene set comprising several classes of proteolytic enzymes was uncovered in the TICs. Accordingly, TICs showed high intra- and extracellular proteolytic activity. Application of a broad range of protease inhibitors to TICs in a colony formation assay reduced anchorage independent growth and had an impact on colony morphology in 3D cell culture assays. We conclude that CD24+CD90+CD45- cells of the MMTV- PyMT mouse model possess an upregulated proteolytic signature which could very well represent a functional hallmark of metastatic TICs from mammary carcinomas., Competing Interests: Conflicts of Interest: None declared.

Carcinomas establish a molecular cross talk between malignant tumor cells and the activated non-malignant cells of the tumor stroma. This cell-cell communication in tumor-stroma interaction includes soluble, secreted proteins that act in a paracrine or autocrine manner. Proteases are crucial factors in tumor-stroma interaction by degrading or truncating secreted bioactive proteins. The cysteine protease cathepsin B is frequently overexpressed in several cancer types, including breast cancer. Its abundance often correlates with poor prognosis. In the murine polyoma virus middle T oncogene (PyMT) breast cancer model, cathepsin B is equally pro-tumorigenic. In this study, we investigate how cathepsin B shapes the secreted proteome of PyMT breast cancers. We employed a novel strategy to harvest tumor interstitial fluid (IF) in combination with chemical stable isotope tagging for quantitative proteomic comparison of IF stemming from PyMT tumors from wild-type mice, mice lacking cathepsin B, and mice over-expressing human cathepsin B. In three biological replicates, we achieve good proteome coverage (∼1700 proteins), with a large content (>70%) of secreted proteins. This characterizes IF as a robust source for the investigation of cancer secretomes. We also identified a large number of shed ectodomains, thus highlighting the importance of tumor-contextual cell surface proteolysis. Furthermore, IF contained >190 proteases and protease inhibitors, which span the entire range of absolute protein abundances; an observation testifying for an important role of proteolysis in tumor-stroma interaction. The cathepsin B genotype consistently affected proteins including alpha-1B-glycoprotein and major urinary proteins 11 and 8 (MUP8). Our study establishes tumor IF as a rich source for the investigation of secreted proteins in tumor biology and sheds light on complex proteolytic networks in the breast cancer secretome., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The lysosomal protease cathepsin L has been reported to cleave various functionally important cytosolic or nuclear proteins. To explain nucleo-cytosolic localization of cathepsin L, it has been hypothesized that skipping of the first start codon during translation initiation results in an N-terminally truncated protein lacking the endoplasmic reticulum-import signal. Here we demonstrate that out-of-frame AUGs prevent translation of truncated cathepsin L in cell culture as well as in a new knock-in mouse model. We further evaluate potential roles of nuclear cathepsin L during early embryonic development. Our analysis reveals normal epiblast development of cathepsin L-deficient embryos, but uncovers a pronounced lysosomal storage phenotype in the extra-embryonic tissue of the visceral endoderm. In conclusion, the phenotypes of cathepsin L deficiency can be fully assigned to lack of canonically targeted cathepsin L, while the biogenesis and functionality of nucleo-cytosolic cathepsin L remain elusive.

The cysteine protease cathepsin B (CTSB) is frequently overexpressed in human breast cancer and correlated with a poor prognosis. Genetic deficiency or pharmacological inhibition of CTSB attenuates tumor growth, invasion and metastasis in mouse models of human cancers. CTSB is expressed in both cancer cells and cells of the tumor stroma, in particular in tumor-associated macrophages (TAM). In order to evaluate the impact of tumor- or stromal cell-derived CTSB on Polyoma Middle T (PyMT)-induced breast cancer progression, we used in vivo and in vitro approaches to induce human CTSB overexpression in PyMT cancer cells or stromal cells alone or in combination. Orthotopic transplantation experiments revealed that CTSB overexpression in cancer cells rather than in the stroma affects PyMT tumor progression. In 3D cultures, primary PyMT tumor cells showed higher extracellular matrix proteolysis and enhanced collective cell invasion when CTSB was overexpressed and proteolytically active. Coculture of PyMT cells with bone marrow-derived macrophages induced a TAM-like macrophage phenotype in vitro, and the presence of such M2-polarized macrophages in 3D cultures enhanced sprouting of tumor spheroids. We employed a doxycycline (DOX)-inducible CTSB expression system to selectively overexpress human CTSB either in cancer cells or in macrophages in 3D cocultures. Tumor spheroid invasiveness was only enhanced when CTSB was overexpressed in cancer cells, whereas CTSB expression in macrophages alone did not further promote invasiveness of tumor spheroids. We conclude that CTSB overexpression in the PyMT mouse model promotes tumor progression not by a stromal effect, but by a direct, cancer cell-inherent mode of action: CTSB overexpression renders the PyMT cancers more invasive by increasing proteolytic extracellular matrix protein degradation fostering collective cell invasion into adjacent tissue.