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Post-lactational involution in the mammary gland is shown to be accomplished by a lysosome-mediated cell death pathway. This pathway is independent of the executioner caspases 3, 6 and 7, and instead relies on Stat3-mediated upregulation of cathepsins. It is well established that lysosomes play an active role during the execution of cell death 1 . A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme 2 , 3 . However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions 4 , 5 , 6 , 7 . Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref.  8 ). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively 1 , 9 , 10 .

Breast cancers are highly heterogeneous and their metastatic potential and response to therapeutic drugs is difficult to predict. A tool that could accurately gauge tumour invasiveness and drug response would provide a valuable addition to the oncologist’s arsenal. We have developed a 3-dimensional (3D) culture model that recapitulates the stromal environment of breast cancers by generating anisotropic (directional) collagen scaffolds seeded with adipocytes and culturing tumour fragments therein. Analysis of tumour cell invasion in the presence of various therapeutic drugs, by immunofluorescence microscopy coupled with an optical clearing technique, demonstrated the utility of this approach in determining both the rate and capacity of tumour cells to migrate through the stroma while shedding light also on the mode of migration. Furthermore, the response of different murine mammary tumour types to chemotherapeutic drugs could be readily quantified.

While genetically engineered mice have made an enormous contribution towards the elucidation of human disease, it has hitherto not been possible to tune up or down the level of expression of any endogenous gene. Here we describe compound genetically modified mice in which expression of the endogenous E2f3 gene may be either reversibly elevated or repressed in adult animals by oral administration of tetracycline. This technology is, in principle, applicable to any endogenous gene, allowing direct determination of both elevated and reduced gene expression in physiological and pathological processes. Applying this switchable technology to the key cell cycle transcription factor E2F3, we demonstrate that elevated levels of E2F3 drive ectopic proliferation in multiple tissues. By contrast, E2F3 repression has minimal impact on tissue proliferation or homeostasis in the majority of contexts due to redundancy of adult function with E2F1 and E2F2. In the absence of E2F1 and E2F2, however, repression of E2F3 elicits profound reduction of proliferation in the hematopoietic compartments that is rapidly lethal in adult animals.

Cysteine cathepsins are proteolytic enzymes that reside in endolysosomal vesicles. Some are expressed constitutively while others are transcriptionally regulated. However, the expression and subcellular localization of cathepsins changes during cancer progression and cathepsins have been shown to be causally involved in various aspects of tumorigenesis including metastasis. The use of mouse models of breast cancer genetically ablated for cathepsin B has shown that both the growth of the primary tumor and the extend of lung metastasis is reduced by the loss of cathepsin B. The role of cathepsins in involution of the mammary gland has received little attention although it is clear that cathepsins are involved in tissue remodeling in the second phase of involution. We discuss here the roles of cathepsins and their endogenous inhibitors in breast tumorigenesis and post-lactational involution.

Key Points A number of alternative cell death programmes, such as necroptosis, lysosomal-mediated programmed cell death (LM-PCD) and autophagy have been established alongside classical apoptosis. These are now known to act both as a backup to apoptosis and also as preferred death pathways in certain cell types. Necroptosis can be triggered by a RIP1- and RIP3-containing complex. This complex can form downstream of death receptors or in the cytosol following stress stimuli. It is tightly regulated by the initiator caspase 8, as well as inhibitors of apoptosis (IAPs) and FLICE-like inhibitory protein (FLIP). Hence, caspase inhibitors, as well as second mitochondria-derived activator of caspases (SMAC) mimetics, are strong sensitizers for necroptosis. LM-PCD, which occurs because of a loss of lysosomal integrity, is frequently seen in cancer cells. This is due to increased metabolism and protein turnover, as well as a reduction of important lysosomal structural proteins. Cancer cells are thus particularly responsive to drugs that target the lysosomes, and drugs ranging from lysosomotropic agents to monoclonal antibodies and microtubule-disrupting agents have all been shown to induce LM-PCD. Autophagy can have both tumour suppressive and tumour-promoting activities. Large-scale autophagy can eventually lead to cell death; however, it is not clear precisely how this type of death is induced. Inhibition and induction of autophagy have both proved to be beneficial under certain circumstances, and the decision as to whether inhibition or activation is preferable is still largely empirical. The alternative pathways of cell death prove to be intricately interconnected with many points of convergence, such as JUN N-terminal kinase (JNK), AMP-activated protein kinase (AMPK) or reactive oxygen species (ROS). Necroptosis has LM-PCD as a frequent downstream occurrence, and an impaired lysosomal compartment affects the ability of autophagosomes to mature. These converging and diverging features are increasingly better understood, leading to a number of targeted approaches aimed at these alternative pathways. Research over the past decade has greatly increased our understanding of non-apoptotic programmed cell death events, such as lysosomal-mediated cell death, necroptosis and cell death with autophagy. This Review discusses converging and diverging features of these pathways with a view to developing new therapeutics for cancer. Evading programmed cell death is one of the hallmarks of cancer. Conversely, inducing cell death by pharmacological means is the basis of almost every non-invasive cancer therapy. Research over the past decade has greatly increased our understanding of non-apoptotic programmed cell death events, such as lysosomal-mediated cell death, necroptosis and cell death with autophagy. It is becoming clear that an intricate effector network connects many of these classical and non-classical death pathways. In this Review, we discuss converging and diverging features of these pathways, as well as attempts to exploit this newly gained knowledge pharmacologically to provide therapeutics for cancer.

Cells with higher levels of Myc proliferate more rapidly and supercompetitively eliminate neighboring cells. Nonetheless, tumor cells in aggressive breast cancers typically exhibit significant and stable heterogeneity in their Myc levels, which correlates with refractoriness to therapy and poor prognosis. This suggests that Myc heterogeneity confers some selective advantage on breast tumor growth and progression. To investigate this, we created a traceable MMTV-Wnt1–driven in vivo chimeric mammary tumor model comprising an admixture of low-Myc– and reversibly switchable high-Myc–expressing clones. We show that such tumors exhibit interclonal mutualism wherein cells with high-Myc expression facilitate tumor growth by promoting protumorigenic stroma yet concomitantly suppress Wnt expression, which renders them dependent for survival on paracrine Wnt provided by low-Myc–expressing clones. To identify any therapeutic vulnerabilities arising from such interdependency, we modeled Myc/Ras/p53/Wnt signaling cross talk as an executable network for low-Myc, for high-Myc clones, and for the 2 together. This executable mechanistic model replicated the observed interdependence of high-Myc and low-Myc clones and predicted a pharmacological vulnerability to coinhibition of COX2 and MEK. This was confirmed experimentally. Our study illustrates the power of executable models in elucidating mechanisms driving tumor heterogeneity and offers an innovative strategy for identifying combination therapies tailored to the oligoclonal landscape of heterogenous tumors.

C‐reactive protein (CRP) is an early‐stage acute phase protein and highly upregulated in response to inflammatory reactions. We recently identified a novel mechanism that leads to a conformational change from the native, functionally relatively inert, pentameric CRP (pCRP) structure to a pentameric CRP intermediate (pCRP*) and ultimately to the monomeric CRP (mCRP) form, both exhibiting highly pro‐inflammatory effects. This transition in the inflammatory profile of CRP is mediated by binding of pCRP to activated/damaged cell membranes via exposed phosphocholine lipid head groups. We designed a tool compound as a low molecular weight CRP inhibitor using the structure of phosphocholine as a template. X‐ray crystallography revealed specific binding to the phosphocholine binding pockets of pCRP. We provide in vitro and in vivo proof‐of‐concept data demonstrating that the low molecular weight tool compound inhibits CRP‐driven exacerbation of local inflammatory responses, while potentially preserving pathogen‐defense functions of CRP. The inhibition of the conformational change generating pro‐inflammatory CRP isoforms via phosphocholine‐mimicking compounds represents a promising, potentially broadly applicable anti‐inflammatory therapy. Synopsis A novel low molecular weight compound C10M was designed to block the PC binding site on pCRP, thereby inhibiting the formation of the pro‐inflammatory isoforms pCRP*/mCRP, and thus showing broad anti‐inflammatory effects in vitro and in vivo . The feasibility of the monovalent approach of PC binding site inhibition was demonstrated directly by X‐ray crystallography. The pro‐inflammatory conformational change of pCRP was blocked by a novel monovalent inhibitor utilizing the PC binding site, leaving the B‐face otherwise accessible. The compound C10M inhibited pCRP*/mCRP‐dependent pro‐inflammatory effects on endothelial cells, monocytes and leukocytes in vitro . pCRP*/mCRP‐driven inflammation in renal ischemia/reperfusion‐injury and VCA hindlimb rejection in vivo was markedly inhibited by C10M. Graphical Abstract A novel low molecular weight compound C10M was designed to block the PC binding site on pCRP, thereby inhibiting the formation of the pro‐inflammatory isoforms pCRP*/mCRP, and thus showing broad anti‐inflammatory effects in vitro and in vivo .

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras -mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC. Colorectal tumors with mutated KRAS and APC require the amino acid transporter SLC7A5 to drive tumorigenesis. Mechanistically, SLC7A5 drives transcriptional and metabolic reprogramming by maintaining intracellular amino acid levels, leading to enhanced protein synthesis.

Lymphotoxin β receptor (LTβR), a member of the TNF receptor superfamily (TNFR-SF), is essential for development and maturation of lymphoid organs. In addition, LTβR activation promotes carcinogenesis by inducing a proinflammatory secretome. Yet, we currently lack a detailed understanding of LTβR signaling. In this study we discovered the linear ubiquitin chain assembly complex (LUBAC) as a previously unrecognized and functionally crucial component of the native LTβR signaling complex (LTβR-SC). Mechanistically, LUBAC-generated linear ubiquitin chains enable recruitment of NEMO, OPTN and A20 to the LTβR-SC, where they act coordinately to regulate the balance between canonical and non-canonical NF-κB pathways. Thus, different from death receptor signaling, where LUBAC prevents inflammation through inhibition of cell death, in LTβR signaling LUBAC is required for inflammatory signaling by enabling canonical and interfering with non-canonical NF-κB activation. This results in a LUBAC-dependent LTβR-driven inflammatory, protumorigenic secretome. Intriguingly, in liver cancer patients with high LTβR expression, high expression of LUBAC correlates with poor prognosis, providing clinical relevance for LUBAC-mediated inflammatory LTβR signaling.