Aude Mallavialle

Cathepsins, the most abundant lysosomal proteases, have key functions in cell maintenance and homeostasis. They are overexpressed and hypersecreted in cancer and associated with poor prognosis. Secreted cathepsins display pro‐tumour activities in the tumour microenvironment and thus represent interesting molecular targets in oncology. Recently, several antibody‐based cancer therapies have targeted the pro‐tumour activity of the extracellular cathepsin pool, altering several cancer hallmarks, but not the intracellular cathepsin levels that are often crucial for cell homeostasis. In this mini‐review, we describe advances in antibodies against extracellular cathepsins in cancer, and their effect on the proteolytic cascade, matrix remodelling, proliferation, and modulation of the anti‐cancer immune response. We also discuss the add‐on value of combination strategies (anti‐cathepsin antibodies with chemotherapy and/or biologics) that make anti‐cathepsin antibodies a new opportunity for disease management.

Introduction: Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. Methods: Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc⁺) or prevent (F1M1-Fc⁻) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc⁺ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. Results: Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc⁺ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc⁻ was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc⁺ displayed higher antitumor activity than F1M1, whereas F1M1-Fc⁻ was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc⁺ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc⁺ antitumor activity, demonstrating their key role. F1M1-Fc⁺ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc⁺ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. Conclusions: F1M1-Fc⁺ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Supplementary Figure 1. b-AP15 and its derivative VLX1570 inhibit USP14 activity in melanoma cells. Supplementary Figure 2. USP14 inhibition irreversibly induces growth inhibition and cytotoxicity in melanoma cells. Supplementary Figure 3. USP14 overexpression reduces the cytotoxic effect of b-AP15 on melanoma cell. Supplementary Figure 4. siRNA-mediated depletion of USP14 reduces melanoma cell growth and focus formation. Supplementary Figure 5. USP14 targeting induces caspase-independent and ROS-dependent cell death. Supplementary Figure 6. USP14 inhibition induces a rapid accumulation of polyubiquitinated proteins and HSP70 in melanoma cells

Fibroblastic reticular cells (FRC) are immunologically specialized myofibroblasts that control the elasticity of the lymph node, in part through their contractile properties. Swelling of tumor-draining lymph nodes is a hallmark of lymphophilic cancers such as cutaneous melanoma. Melanoma displays high intratumoral heterogeneity with the coexistence of melanoma cells with variable differentiation phenotypes from melanocytic to dedifferentiated states. Factors secreted by melanoma cells promote premetastatic lymph node reprograming and tumor spreading. Elucidating the impact of the melanoma secretome on FRC could help identify approaches to prevent metastasis. Here we show that melanocytic and dedifferentiated melanoma cells differentially impact the FRC contractile phenotype. Factors secreted by dedifferentiated cells, but not by melanocytic cells, strongly inhibited actomyosin-dependent contractile forces of FRC by decreasing the activity of the RHOA–RHO–kinase (ROCK) pathway and the mechano-responsive transcriptional coactivator Yes1 associated transcriptional regulator (YAP). Transcriptional profiling and biochemical analyses indicated that actomyosin cytoskeleton relaxation in FRC is driven by inhibition of the JAK1-STAT3 pathway. This FRC relaxation was associated with increased FRC proliferation and activation and with elevated tumor invasion in vitro. The secretome of dedifferentiated melanoma cells also modulated the biomechanical properties of distant lymph node in premetastatic mouse models. Finally, IL1 produced by dedifferentiated cells was involved in the inhibition of FRC contractility. These data highlight the role of the JAK1-STAT3 and YAP pathways in spontaneous contractility of resting FRC. They also suggest that dedifferentiated melanoma cells specifically target FRC biomechanical properties to favor tumor spreading in the premetastatic lymph node niche. Targeting this remote communication could be an effective strategy to prevent metastatic spread of the disease.Significance:Communication between dedifferentiated melanoma cells and lymph node fibroblasts reprograms the biomechanical properties of the premetastatic lymph node niche to promote tumor invasion.See related commentary by Lund, p. 1692

Supplementary Figure from Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3–Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells

Supplementary Figure from Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3–Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells

Supplementary Data from Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3–Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells

Supplementary Figure from Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3–Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells

Supplementary Figure from Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3–Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells