A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

Colon tumour cell death causes mTOR dependence by paracrine P2X4 stimulation.

Solid cancers exhibit a dynamic balance between cell death and proliferation ensuring continuous tumour maintenance and growth1,2. Increasing evidence links enhanced cancer cell apoptosis to paracrine activation of cells in the tumour microenvironment initiating tissue repair programs that support tumour growth3,4, yet the direct effects of dying cancer cells on neighbouring tumour epithelia and how this paracrine effect potentially contributes to therapy resistance are unclear. Here we demonstrate that chemotherapy-induced tumour cell death in patient-derived colorectal tumour organoids causes ATP release triggering P2X4 (also known as P2RX4) to mediate an mTOR-dependent pro-survival program in neighbouring cancer cells, which renders surviving tumour epithelia sensitive to mTOR inhibition. The induced mTOR addiction in persisting epithelial cells is due to elevated production of reactive oxygen species and subsequent increased DNA damage in response to the death of neighbouring cells. Accordingly, inhibition of the P2X4 receptor or direct mTOR blockade prevents induction of S6 phosphorylation and synergizes with chemotherapy to cause massive cell death induced by reactive oxygen species and marked tumour regression that is not seen when individually applied. Conversely, scavenging of reactive oxygen species prevents cancer cells from becoming reliant on mTOR activation. Collectively, our findings show that dying cancer cells establish a new dependency on anti-apoptotic programs in their surviving neighbours, thereby creating an opportunity for combination therapy in P2X4-expressing epithelial tumours.

Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade.

OBJECTIVE: Investigating the effect of ferroptosis in the tumour microenvironment to identify combinatory therapy for liver cancer treatment. DESIGN: Glutathione peroxidase 4 (GPx4), which is considered the master regulator of ferroptosis, was genetically altered in murine models for hepatocellular carcinoma (HCC) and colorectal cancer (CRC) to analyse the effect of ferroptosis on tumour cells and the immune tumour microenvironment. The findings served as foundation for the identification of additional targets for combine therapy with ferroptotic inducer in the treatment of HCC and liver metastasis. RESULTS: Surprisingly, hepatocyte-restricted GPx4 loss does not suppress hepatocellular tumourigenesis. Instead, GPx4-associated ferroptotic hepatocyte death causes a tumour suppressive immune response characterised by a CXCL10-dependent infiltration of cytotoxic CD8+ T cells that is counterbalanced by PD-L1 upregulation on tumour cells as well as by a marked HMGB1-mediated myeloid derived suppressor cell (MDSC) infiltration. Blocking PD-1 or HMGB1 unleashes T cell activation and prolongs survival of mice with Gpx4-deficient liver tumours. A triple combination of the ferroptosis inducing natural compound withaferin A, the CXCR2 inhibitor SB225002 and α-PD-1 greatly improves survival of wild-type mice with liver tumours. In contrast, the same combination does not affect tumour growth of subcutaneously grown CRC organoids, while it decreases their metastatic growth in liver. CONCLUSION: Our data highlight a context-specific ferroptosis-induced immune response that could be therapeutically exploited for the treatment of primary liver tumours and liver metastases.

Depression, Anxiety, Stress, and Health-Related Quality of Life Among Patients With Medication Overuse Headache in a Tertiary Headache Center: A Cross-Sectional Study.

OBJECTIVE: The investigators examined the association of patient-related and headache-related parameters and the effect of medication overuse headache (MOH); the occurrence of depression, anxiety, and stress; and the importance of different domains of health-related quality of life in these associations. METHODS: Eighty-three patients (women, N=72, men, N=11; mean age, 40.54 years, SD=11.58), who were first diagnosed with MOH during the study period were included in the analyses. The Headache Impact Test-6 (HIT-6), the 36-item Short-Form Survey (SF-36) Questionnaire for quality of life, and the Depression Anxiety Stress Scales were used. RESULTS: The findings revealed mild depression, moderate anxiety, and stress, as well as changes in all examined health domains, in the study patients (p<0.05). Risk factors were identified for higher HIT-6 scores (role functioning/physical functioning [odds ratio=0.977, p=0.024] and social functioning [odds ratio=0.963, p=0.032]); for depression (emotional well-being [odds ratio=0.928, p=0.007], social functioning [odds ratio=0.950, p=0.009], and the presence of comorbidity [odds ratio=5.417, p=0.013]); for anxiety (age [odds ratio=1.091, p=0.007], MOH duration [odds ratio=1.422, p=0.047], emotional well-being [odds ratio=0.933, p=0.012], and social functioning [odds ratio=0.943, p=0.001]); and for stress (emotional well-being [odds ratio=0.902, p<0.001]). CONCLUSIONS: MOH has a significant negative impact on the personal, family, and social life of patients and is associated with depression, anxiety, and stress. Patients' age, duration of MOH, presence of comorbidities, and adverse effects of physical, emotional, and social dysfunction are particularly important contributors to the negative effects of MOH.

STAT3 activation through IL-6/IL-11 in cancer-associated fibroblasts promotes colorectal tumour development and correlates with poor prognosis.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) influence the tumour microenvironment and tumour growth. However, the role of CAFs in colorectal cancer (CRC) development is incompletely understood. DESIGN: We quantified phosphorylation of STAT3 (pSTAT3) expression in CAFs of human colon cancer tissue using a tissue microarray (TMA) of 375 patients, immunofluorescence staining and digital pathology. To investigate the functional role of CAFs in CRC, we took advantage of two murine models of colorectal neoplasia and advanced imaging technologies. In loss-of-function and gain-of-function experiments, using genetically modified mice with collagen type VI (COLVI)-specific signal transducer and activator of transcription 3 (STAT3) targeting, we evaluated STAT3 signalling in fibroblasts during colorectal tumour development. We performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblast subpopulations (COLVI+ vs COLVI-) on STAT3 activation (IL-6 vs IL-11). RESULTS: The analysis of pSTAT3 expression in CAFs of human TMAs revealed a negative correlation of increased stromal pSTAT3 expression with the survival of colon cancer patients. In the loss-of-function and gain-of-function approach, we found a critical role of STAT3 activation in fibroblasts in driving colorectal tumourigenesis in vivo. With different imaging technologies, we detected an expansion of activated fibroblasts in colorectal neoplasias. Comparative gene expression profiling of fibroblast subpopulations on STAT3 activation revealed the regulation of transcriptional patterns associated with angiogenesis. Finally, the blockade of proangiogenic signalling significantly reduced colorectal tumour growth in mice with constitutive STAT3 activation in COLVI+ fibroblasts. CONCLUSION: Altogether our work demonstrates a critical role of STAT3 activation in CAFs in CRC development.

T-helper-1-cell cytokines drive cancer into senescence.

Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

Single-cell deconvolution reveals high lineage- and location-dependent heterogeneity in mesenchymal multivisceral stage 4 colorectal cancer.

Metastasized colorectal cancer (CRC) is associated with a poor prognosis and rapid disease progression. Besides hepatic metastasis, peritoneal carcinomatosis is the major cause of death in Union for International Cancer Control (UICC) stage IV CRC patients. Insights into differential site-specific reconstitution of tumor cells and the corresponding tumor microenvironment are still missing. Here, we analyzed the transcriptome of single cells derived from murine multivisceral CRC and delineated the intermetastatic cellular heterogeneity regarding tumor epithelium, stroma, and immune cells. Interestingly, we found an intercellular site-specific network of cancer-associated fibroblasts and tumor epithelium during peritoneal metastasis as well as an autologous feed-forward loop in cancer stem cells. We furthermore deciphered a metastatic dysfunctional adaptive immunity by a loss of B cell-dependent antigen presentation and consecutive effector T cell exhaustion. Furthermore, we demonstrated major similarities of this murine metastatic CRC model with human disease and - based on the results of our analysis - provided an auspicious site-specific immunomodulatory treatment approach for stage IV CRC by intraperitoneal checkpoint inhibition.

Image-guided radiotherapy in an orthotopic mouse model of rectal cancer.

Radiobiology research in rectal cancer has been limited to cell lines, patient-derived organoids (PDOs), or xenografts. Here, we describe a protocol which recapitulates more efficiently the complex contributions of the tumor microenvironment. This approach establishes a preclinical mouse model of rectal cancer by intrarectal transplantation of genetically modified organoids into immunocompetent mice followed by precise image-guided radiotherapy (IGRT) of organoid-induced tumors. This model represents a useful platform to study the cellular and molecular determinants of therapy resistance in rectal cancer. For complete details on the use and execution of this protocol, please refer to Nicolas et al. (2022).

Inflammatory fibroblasts mediate resistance to neoadjuvant therapy in rectal cancer.

Standard cancer therapy targets tumor cells without considering possible damage on the tumor microenvironment that could impair therapy response. In rectal cancer patients we find that inflammatory cancer-associated fibroblasts (iCAFs) are associated with poor chemoradiotherapy response. Employing a murine rectal cancer model or patient-derived tumor organoids and primary stroma cells, we show that, upon irradiation, interleukin-1α (IL-1α) not only polarizes cancer-associated fibroblasts toward the inflammatory phenotype but also triggers oxidative DNA damage, thereby predisposing iCAFs to p53-mediated therapy-induced senescence, which in turn results in chemoradiotherapy resistance and disease progression. Consistently, IL-1 inhibition, prevention of iCAFs senescence, or senolytic therapy sensitizes mice to irradiation, while lower IL-1 receptor antagonist serum levels in rectal patients correlate with poor prognosis. Collectively, we unravel a critical role for iCAFs in rectal cancer therapy resistance and identify IL-1 signaling as an attractive target for stroma-repolarization and prevention of cancer-associated fibroblasts senescence.

Facial Emotion Recognition and Persecutory Ideation in Paranoid Schizophrenia.

The recognition of facial signals has a crucial role in social interaction. It is well known that people suffering from paranoid schizophrenia have problems in the social domain, predominantly related to misinterpreting the intentions, emotions, and actions of others. The aim of this study was to examine whether there are differences in facial emotion recognition between people with paranoid schizophrenia and healthy controls. In addition, we examined the correlation between facial emotion recognition and the expression of persecutory ideation in people suffering from paranoid schizophrenia. The study involved 60 participants, 30 of whom suffered from paranoid schizophrenia and 30 healthy controls, equalized by gender, age, and education. The following instruments were used: Japanese and Caucasian Facial Expressions of Emotion and Neutral Faces and the Persecutory Ideation Questionnaire. Compared with the controls, people suffering from paranoid schizophrenia were significantly less accurate in recognizing the following emotions: surprise, contempt, sadness, disgust, and emotionally neutral faces. Since the attribution of emotions to emotionally neutral faces is an important finding that could be linked with the social (dis)functionality of people suffering from paranoid schizophrenia, we analyzed and compared the wrong answers given by the two groups and found some differences between them. The results show that persecutory ideation has a statistically significant negative correlation with the successful recognition of emotionally neutral faces. All of the findings lead to the conclusion that paranoid schizophrenia, and within it the existence of persecutory ideation, leads to problems in recognizing the basic facial signals that form the foundation of everyday social interaction.

AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer.

Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack of animal models that faithfully recapitulate the different molecular subtypes impedes adequate preclinical testing of stratified therapeutic concepts. Here, we demonstrate that constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis in Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with the carcinogen azoxymethane. Gene-expression profiling indicates that Trp53ΔIECAktE17K tumors resemble the human mesenchymal colorectal cancer subtype (CMS4), which is characterized by the poorest survival rate among the four CMSs. Trp53ΔIECAktE17K tumor cells are characterized by Notch3 up-regulation, and treatment of Trp53ΔIECAktE17K mice with a NOTCH3-inhibiting antibody reduces invasion and metastasis. In CRC patients, NOTCH3 expression correlates positively with tumor grading and the presence of lymph node as well as distant metastases and is specifically up-regulated in CMS4 tumors. Therefore, we suggest NOTCH3 as a putative target for advanced CMS4 CRC patients.

Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.

Myeloid Cell-Derived Reactive Oxygen Species Induce Epithelial Mutagenesis.

Increased oxidative stress has been suggested to initiate and promote tumorigenesis by inducing DNA damage and to suppress tumor development by triggering apoptosis and senescence. The contribution of individual cell types in the tumor microenvironment to these contrasting effects remains poorly understood. We provide evidence that during intestinal tumorigenesis, myeloid cell-derived H2O2 triggers genome-wide DNA mutations in intestinal epithelial cells to stimulate invasive growth. Moreover, increased reactive oxygen species (ROS) production in myeloid cells initiates tumor growth in various organs also in the absence of a carcinogen challenge in a paracrine manner. Our data identify an intricate crosstalk between myeloid cell-derived ROS molecules, oxidative DNA damage, and tumor necrosis factor α-mediated signaling to orchestrate a tumor-promoting microenvironment causing invasive cancer.

Inflammation and cancer: tissue regeneration gone awry.

Unresolved chronic inflammation is implicated in all stages of cancer development and an inflammatory tumor microenvironment is considered a hallmark of cancer. Signaling pathways involved in normal tissue regeneration and repair are dysregulated both in chronic inflammation and cancer. Here, we review some of the recently identified signaling cascades and unexpected functions of stromal cells that affect both tissue regeneration and tumorigenesis in colon and pancreatic cancer, which may pave the way for the development of novel therapeutic strategies.

In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer.

In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.

Inhibiting the HSP90 chaperone destabilizes macrophage migration inhibitory factor and thereby inhibits breast tumor progression.

Intracellular macrophage migration inhibitory factor (MIF) often becomes stabilized in human cancer cells. MIF can promote tumor cell survival, and elevated MIF protein correlates with tumor aggressiveness and poor prognosis. However, the molecular mechanism facilitating MIF stabilization in tumors is not understood. We show that the tumor-activated HSP90 chaperone complex protects MIF from degradation. Pharmacological inhibition of HSP90 activity, or siRNA-mediated knockdown of HSP90 or HDAC6, destabilizes MIF in a variety of human cancer cells. The HSP90-associated E3 ubiquitin ligase CHIP mediates the ensuing proteasome-dependent MIF degradation. Cancer cells contain constitutive endogenous MIF-HSP90 complexes. siRNA-mediated MIF knockdown inhibits proliferation and triggers apoptosis of cultured human cancer cells, whereas HSP90 inhibitor-induced apoptosis is overridden by ectopic MIF expression. In the ErbB2 transgenic model of human HER2-positive breast cancer, genetic ablation of MIF delays tumor progression and prolongs overall survival of mice. Systemic treatment with the HSP90 inhibitor 17AAG reduces MIF expression and blocks growth of MIF-expressing, but not MIF-deficient, tumors. Together, these findings identify MIF as a novel HSP90 client and suggest that HSP90 inhibitors inhibit ErbB2-driven breast tumor growth at least in part by destabilizing MIF.