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.

Loss of SUV420H2-Dependent Chromatin Compaction Drives Right-Sided Colon Cancer Progression.

BACKGROUND & AIMS: Epigenetic processes regulating gene expression contribute markedly to epithelial cell plasticity in colorectal carcinogenesis. The lysine methyltransferase SUV420H2 comprises an important regulator of epithelial plasticity and is primarily responsible for trimethylation of H4K20 (H4K20me3). Loss of H4K20me3 has been suggested as a hallmark of human cancer due to its interaction with DNMT1. However, the role of Suv4-20h2 in colorectal cancer is unknown. METHODS: We examined the alterations in histone modifications in patient-derived colorectal cancer organoids. Patient-derived colorectal cancer organoids and mouse intestinal organoids were genetically manipulated for functional studies in patient-derived xenograft and orthotopic transplantation. Gene expression profiling, micrococcal nuclease assay, and chromatin immunoprecipitation were performed to understand epigenetic regulation of chromatin states and gene expression in patient-derived and mouse intestinal organoids. RESULTS: We found that reduced H4K20me3 levels occurred predominantly in right-sided patient-derived colorectal cancer organoids, which were associated with increased chromatin accessibility. Re-compaction of chromatin by methylstat, a histone demethylase inhibitor, resulted in reduced growth selectively in subcutaneously grown tumors derived from right-sided cancers. Using mouse intestinal organoids, we confirmed that Suv4-20h2-mediated H4K20me3 is required for maintaining heterochromatin compaction and to prevent R-loop formation. Cross-species comparison of Suv4-20h2-depleted murine organoids with right-sided colorectal cancer organoids revealed a large overlap of gene signatures involved in chromatin silencing, DNA methylation, and stemness/Wnt signaling. CONCLUSIONS: Loss of Suv4-20h2-mediated H4K20me3 drives right-sided colorectal tumorigenesis through an epigenetically controlled mechanism of chromatin compaction. Our findings unravel a conceptually novel approach for subtype-specific therapy of this aggressive form of colorectal cancer.

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.

The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer.

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.

Fructose stimulated de novo lipogenesis is promoted by inflammation.

Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.

Therapeutic Ablation of Gain-of-Function Mutant p53 in Colorectal Cancer Inhibits Stat3-Mediated Tumor Growth and Invasion.

Over half of colorectal cancers (CRCs) harbor TP53 missense mutations (mutp53). We show that the most common mutp53 allele R248Q (p53Q) exerts gain of function (GOF) and creates tumor dependence in mouse CRC models. mutp53 protein binds Stat3 and enhances activating Stat3 phosphorylation by displacing the phosphatase SHP2. Ablation of the p53Q allele suppressed Jak2/Stat3 signaling, growth, and invasiveness of established, mutp53-driven tumors. Treating tumor-bearing mice with an HSP90 inhibitor suppressed mutp53 levels and tumor growth. Importantly, human CRCs with stabilized mutp53 exhibit enhanced Jak2/Stat3 signaling and are associated with poorer patient survival. Cancers with TP53R248Q/W are associated with a higher patient death risk than are those having nonR248 mutp53. These findings identify GOF mutp53 as a therapeutic target in CRC.

Colorectal Tumors Require NUAK1 for Protection from Oxidative Stress.

Exploiting oxidative stress has recently emerged as a plausible strategy for treatment of human cancer, and antioxidant defenses are implicated in resistance to chemotherapy and radiotherapy. Targeted suppression of antioxidant defenses could thus broadly improve therapeutic outcomes. Here, we identify the AMPK-related kinase NUAK1 as a key component of the antioxidant stress response pathway and reveal a specific requirement for this role of NUAK1 in colorectal cancer. We show that NUAK1 is activated by oxidative stress and that this activation is required to facilitate nuclear import of the antioxidant master regulator NRF2: Activation of NUAK1 coordinates PP1ß inhibition with AKT activation in order to suppress GSK3ß-dependent inhibition of NRF2 nuclear import. Deletion of NUAK1 suppresses formation of colorectal tumors, whereas acute depletion of NUAK1 induces regression of preexisting autochthonous tumors. Importantly, elevated expression of NUAK1 in human colorectal cancer is associated with more aggressive disease and reduced overall survival.Significance: This work identifies NUAK1 as a key facilitator of the adaptive antioxidant response that is associated with aggressive disease and worse outcome in human colorectal cancer. Our data suggest that transient NUAK1 inhibition may provide a safe and effective means for treatment of human colorectal cancer via disruption of intrinsic antioxidant defenses. Cancer Discov; 8(5); 632-47. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Corrigendum: Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

This corrects the article DOI: 10.1038/nature22056.

Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

The non-essential amino acids serine and glycine are used in multiple anabolic processes that support cancer cell growth and proliferation (reviewed in ref. 1). While some cancer cells upregulate de novo serine synthesis, many others rely on exogenous serine for optimal growth. Restriction of dietary serine and glycine can reduce tumour growth in xenograft and allograft models. Here we show that this observation translates into more clinically relevant autochthonous tumours in genetically engineered mouse models of intestinal cancer (driven by Apc inactivation) or lymphoma (driven by Myc activation). The increased survival following dietary restriction of serine and glycine in these models was further improved by antagonizing the anti-oxidant response. Disruption of mitochondrial oxidative phosphorylation (using biguanides) led to a complex response that could improve or impede the anti-tumour effect of serine and glycine starvation. Notably, Kras-driven mouse models of pancreatic and intestinal cancers were less responsive to depletion of serine and glycine, reflecting an ability of activated Kras to increase the expression of enzymes that are part of the serine synthesis pathway and thus promote de novo serine synthesis.

Opposing effects of TIGAR- and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine.

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation.

E-cadherin can limit the transforming properties of activating ß-catenin mutations.

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in ß-catenin (CTNNB1). We have compared the dynamics and the potency of ß-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of ß-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of ß-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of ß-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:ß-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of ß-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of ß-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated ß-catenin.

PROX1 promotes metabolic adaptation and fuels outgrowth of Wnt(high) metastatic colon cancer cells.

The Wnt pathway is abnormally activated in the majority of colorectal cancers, and significant knowledge has been gained in understanding its role in tumor initiation. However, the mechanisms of metastatic outgrowth in colorectal cancer remain a major challenge. We report that autophagy-dependent metabolic adaptation and survival of metastatic colorectal cancer cells is regulated by the target of oncogenic Wnt signaling, homeobox transcription factor PROX1, expressed by a subpopulation of colon cancer progenitor/stem cells. We identify direct PROX1 target genes and show that repression of a pro-apoptotic member of the BCL2 family, BCL2L15, is important for survival of PROX1(+) cells under metabolic stress. PROX1 inactivation after the establishment of metastases prevented further growth of lesions. Furthermore, autophagy inhibition efficiently targeted metastatic PROX1(+) cells, suggesting a potential therapeutic approach. These data identify PROX1 as a key regulator of the transcriptional network contributing to metastases outgrowth in colorectal cancer.

MYC-induced epigenetic activation of GATA4 in lung adenocarcinoma.

Human lung cancer is a disease with high incidence and accounts for most cancer-related deaths in both men and women. Metastasis is a common event in non-small cell lung carcinoma (NSCLC), diminishing the survival chance of the patients with this type of tumor. It has been shown that MYC is involved in the development of metastasis from NSCLC, but the mechanism underlying this switch remained to be identified. Here, we focus on GATA4 as a MYC target in the development of metastasis with origin in lung adenocarcinoma, the most common type of NSCLC. Epigenetic alterations at the GATA4 promoter level were observed after MYC expression in lung adenocarcinoma in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. Our results thus identify a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in lung adenocarcinoma, suggesting novel potential targets for the development of antimetastatic therapy.

E-cadherin controls bronchiolar progenitor cells and onset of preneoplastic lesions in mice.

Although progenitor cells of the conducting airway have been spatially localized and some insights have been gained regarding their molecular phenotype, relatively little is known about the mechanisms regulating their maintenance, activation, and differentiation. This study investigates the potential roles of E-cadherin in mouse Clara cells, as these cells were shown to represent the progenitor/stem cells of the conducting airways and have been implicated as the cell of origin of human non-small cell lung cancer. Postnatal inactivation of E-cadherin affected Clara cell differentiation and compromised airway regeneration under injury conditions. In steady-state adult lung, overexpression of the dominant negative E-cadherin led to an expansion of the bronchiolar stem cells and decreased differentiation concomitant with canonical Wnt signaling activation. Expansion of the bronchiolar stem cell pool was associated with an incessant proliferation of neuroepithelial body.associated Clara cells that ultimately gave rise to bronchiolar hyperplasia. Despite progressive hyperplasia, only a minority of the mice developed pulmonary solid tumors, suggesting that the loss of E-cadherin function leads to tumor formation when additional mutations are sustained. The present study reveals that E-cadherin plays a critical role in the regulation of proliferation and homeostasis of the epithelial cells lining the conducting airways.

Conditional expression of oncogenic C-RAF in mouse pulmonary epithelial cells reveals differential tumorigenesis and induction of autophagy leading to tumor regression.

Here we describe a novel conditional mouse lung tumor model for investigation of the pathogenesis of human lung cancer. On the basis of the frequent involvement of the Ras-RAF-MEK-ERK signaling pathway in human non-small cell lung carcinoma (NSCLC), we have explored the target cell availability, reversibility, and cell type specificity of transformation by oncogenic C-RAF. Targeting expression to alveolar type II cells or to Clara cells, the two likely precursors of human NSCLC, revealed differential tumorigenicity between these cells. Whereas expression of oncogenic C-RAF in alveolar type II cells readily induced multifocal macroscopic lung tumors independent of the developmental state, few tumors with type II pneumocytes features and incomplete penetrance were found when targeted to Clara cells. Induced tumors did not progress and were strictly dependent on the initiating oncogene. Deinduction of mice resulted in tumor regression due to autophagy rather than apoptosis. Induction of autophagic cell death in regressing lung tumors suggests the use of autophagy enhancers as a treatment choice for patients with NSCLC.

Expression of B-RAF V600E in type II pneumocytes causes abnormalities in alveolar formation, airspace enlargement and tumor formation in mice.

Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Perturbations of these cascades have severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of human tumorigenesis, where B-RAF V600E has been identified as the prevalent mutant. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. The inflammatory cell infiltration did not precede the formation of the lung lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation.

Lin9, a subunit of the mammalian DREAM complex, is essential for embryonic development, for survival of adult mice, and for tumor suppression.

The retinoblastoma tumor suppressor protein (pRB) and related p107 and p130 "pocket proteins" function together with the E2F transcription factors to repress gene expression during the cell cycle and development. Recent biochemical studies have identified the multisubunit DREAM pocket protein complexes in Drosophila melanogaster and Caenorhabditis elegans in regulating developmental gene repression. Although a conserved DREAM complex has also been identified in mammalian cells, its physiological function in vivo has not been determined. Here we addressed this question by targeting Lin9, a conserved core subunit of DREAM. We found that LIN9 is essential for early embryonic development and for viability of adult mice. Loss of Lin9 abolishes proliferation and leads to multiple defects in mitosis and cytokinesis because of its requirement for the expression of a large set of mitotic genes, such as Plk1, Aurora A, and Kif20a. While Lin9 heterozygous mice are healthy and normal, they are more susceptible to lung tumorigenesis induced by oncogenic c-Raf than wild-type mice. Together these experiments provide the first direct genetic evidence for the role of LIN9 in development and mitotic gene regulation and they suggest that it may function as a haploinsufficient tumor suppressor.

MYC is a metastasis gene for non-small-cell lung cancer.

BACKGROUND: Metastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis. METHODOLOGY/PRINCIPAL FINDINGS: Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process. CONCLUSIONS/SIGNIFICANCE: Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development.

Polycomb group protein Bmi1 is required for growth of RAF driven non-small-cell lung cancer.

BACKGROUND: We have previously described a RAF oncogene driven transgenic mouse model for non small cell lung cancer (NSCLC). Here we examine whether tumor initiation and growth requires the stem cell self-renewal factor Bmi1. PRINCIPAL FINDINGS: In order to evaluate Bmi1 function in NSCLC two founder lines that differ in incidence and latency of tumor formation were compared. Ablation of Bmi1 expression in both lines had a dramatically decreased tumor growth. As the line with shorter latency matched the life span of Bmi1 knock out mice, these mice were chosen for further study. The absence of Bmi1 did not decrease the number of tumor initiation in these mice as only the size and not the number of tumors decreased. Reduction in tumor growth resulted from an increase in cell death and decrease in cell cycle progression that corresponded with up-regulation of the p16(INK4a) and p19(ARF). SIGNIFICANCE: The data identifies Bmi1 as an important factor for expansion but not initiation of RAF driven NSCLC.

Oncogene-induced plasticity and cancer stem cells.

The cancer stem cell hypothesis is an evolving concept of oncogenesis that has recently gained wide acceptance. In its simplest form this hypothesis suggests that many if not all tumors arise by consecutive genetic changes in a small subpopulation of cells termed cancer stem cells. These cells have the capacity to sustain tumor growth and are defined by three features: self-renewal, differentiation into the cell types of the original cancer and potent tumor formation. The definition of a cancer stem cell does not necessarily imply its origin from a stem, progenitor or differentiated cell. Hence, the term tumor or cancer initiating cell is often used instead to avoid any implications. Here, we propose a model suggesting that tumor cells progressively acquire stem cell properties as a consequence of oncogene-induced plasticity. The basis of our proposal are data from several experimental in vitro and in vivo models demonstrating reprogramming events triggered by specific combinations of oncogenes. These oncogene combinations not only induce cell lineage switches but also drive the reversal of ontogeny within cell lineages during tumor progression to metastasis. In this perspective article we will summarize the experimental evidence that illustrates our concept and discuss its implications for tumor formation and tumor therapy.