KRAS allelic imbalance drives tumour initiation yet suppresses metastasis in colorectal cancer in vivo.

Oncogenic KRAS mutations are well-described functionally and are known to drive tumorigenesis. Recent reports describe a significant prevalence of KRAS allelic imbalances or gene dosage changes in human cancers, including loss of the wild-type allele in KRAS mutant cancers. However, the role of wild-type KRAS in tumorigenesis and therapeutic response remains elusive. We report an in vivo murine model of colorectal cancer featuring deletion of wild-type Kras in the context of oncogenic Kras. Deletion of wild-type Kras exacerbates oncogenic KRAS signalling through MAPK and thus drives tumour initiation. Absence of wild-type Kras potentiates the oncogenic effect of KRASG12D, while incidentally inducing sensitivity to inhibition of MEK1/2. Importantly, loss of the wild-type allele in aggressive models of KRASG12D-driven CRC significantly alters tumour progression, and suppresses metastasis through modulation of the immune microenvironment. This study highlights the critical role for wild-type Kras upon tumour initiation, progression and therapeutic response in Kras mutant CRC.

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.

Cyclocreatine Suppresses Creatine Metabolism and Impairs Prostate Cancer Progression.

Prostate cancer is the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of the tumor suppressors PTEN and Sprouty2 (SPRY2), we identified enhanced creatine metabolism as a central component of progressive disease. Creatine treatment was associated with enhanced cellular basal respiration in vitro and increased tumor cell proliferation in vivo. Stable isotope tracing revealed that intracellular levels of creatine in prostate cancer cells are predominantly dictated by exogenous availability rather than by de novo synthesis from arginine. Genetic silencing of creatine transporter SLC6A8 depleted intracellular creatine levels and reduced the colony-forming capacity of human prostate cancer cells. Accordingly, in vitro treatment of prostate cancer cells with cyclocreatine, a creatine analog, dramatically reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine and suppressed proliferation. Supplementation with cyclocreatine impaired cancer progression in the PTEN- and SPRY2-deficient prostate cancer GEMMs and in a xenograft liver metastasis model. Collectively, these results identify a metabolic vulnerability in prostate cancer and demonstrate a rational therapeutic strategy to exploit this vulnerability to impede tumor progression. SIGNIFICANCE: Enhanced creatine uptake drives prostate cancer progression and confers a metabolic vulnerability to treatment with the creatine analog cyclocreatine.

CXCR2 inhibition enables NASH-HCC immunotherapy.

OBJECTIVE: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DESIGN: Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. RESULTS: Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. CONCLUSION: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer.

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane-associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC.

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

2,4-dienoyl-CoA reductase regulates lipid homeostasis in treatment-resistant prostate cancer.

Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance.

Modulation of macrophage antitumor potential by apoptotic lymphoma cells.

In aggressive non-Hodgkin's lymphoma (NHL), constitutive apoptosis of a proportion of the tumor cell population can promote net tumor growth. This is associated with the accumulation of tumor-associated macrophages (TAMs) that clear apoptotic cells and exhibit pro-oncogenic transcriptional activation profiles characteristic of reparatory, anti-inflammatory and angiogenic programs. Here we consider further the activation status of these TAMs. We compare their transcriptomic profile with that of a range of other macrophage types from various tissues noting especially their expression of classically activated (IFN-γ and LPS) gene clusters - typically antitumor - in addition to their previously described protumor phenotype. To understand the impact of apoptotic cells on the macrophage activation state, we cocultured apoptotic lymphoma cells with classically activated macrophages (M(IFN-γ/LPS), also known as M1, macrophages). Although untreated and M(IFN-γ/LPS) macrophages were able to bind apoptotic lymphoma cells equally well, M(IFN-γ/LPS) macrophages displayed enhanced ability to phagocytose them. We found that direct exposure of M(IFN-γ/LPS) macrophages to apoptotic lymphoma cells caused switching towards a protumor activation state (often referred to as M2-like) with concomitant inhibition of antitumor activity that was a characteristic feature of M(IFN-γ/LPS) macrophages. Indeed, M(IFN-γ/LPS) macrophages exposed to apoptotic lymphoma cells displayed increased lymphoma growth-promoting activities. Antilymphoma activity by M(IFN-γ/LPS) macrophages was mediated, in part, by galectin-3, a pleiotropic glycoprotein involved in apoptotic cell clearance that is strongly expressed by lymphoma TAMs but not lymphoma cells. Intriguingly, aggressive lymphoma growth was markedly impaired in mice deficient in galectin-3, suggesting either that host galectin-3-mediated antilymphoma activity is required to sustain net tumor growth or that additional functions of galectin-3 drive key oncogenic mechanisms in NHL. These findings have important implications for anticancer therapeutic approaches aimed at polarizing macrophages towards an antitumor state and identify galectin-3 as a potentially important novel target in aggressive NHL.

Microenvironmental Effects of Cell Death in Malignant Disease.

Although apoptosis is well recognized as a cell death program with clear anticancer roles, accumulating evidence linking apoptosis with tissue repair and regeneration indicates that its relationship with malignant disease is more complex than previously thought. Here we review how the responses of neighboring cells in the microenvironment of apoptotic tumor cells may contribute to the cell birth/cell death disequilibrium that provides the basis for cancerous tissue emergence and growth. We describe the bioactive properties of apoptotic cells and consider, in particular, how apoptosis of tumor cells can engender a range of responses including pro-oncogenic signals having proliferative, angiogenic, reparatory, and immunosuppressive features. Drawing on the parallels between wound healing, tissue regeneration and cancer, we propose the concept of the "onco-regenerative niche," a cell death-driven generic network of tissue repair and regenerative mechanisms that are hijacked in cancer. Finally, we consider how the responses to cell death in tumors can be targeted to provide more effective and long-lasting therapies.

Oncogenic properties of apoptotic tumor cells in aggressive B cell lymphoma.

BACKGROUND: Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. RESULTS: Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased "in situ transcriptomics" analysis-gene expression profiling of laser-captured TAMs to establish their activation signature in situ-we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. CONCLUSIONS: In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy.

CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis.

Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase- and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved.

Differential signalling during B-cell maturation.

The molecular mechanism by which the antigen receptors (BCR) on B cells can elicit differential maturation state-specific responses is one of the central problems in B-cell differentiation yet to be resolved. Indeed, many of the early signalling events detected following BCR ligation, such as activation of protein tyrosine kinases (PTK), phospholipase C (PLC), phosphoinositide-3-kinase (PI 3K), protein kinase C (PKC) and the RasMAPK (mitogen activating protein kinase) signalling cascades are observed throughout B-cell maturation. However, it is becoming clear that the differential functional responses of these BCR-coupled signals observed during B-cell maturation are dependent on a number of parameters including signal strength and duration, subcellular localisation of the signal, maturation-restricted expression of downstream signalling effector elements/isoforms and modulation of signal by co-receptors. Thus, the combined signature of BCR signalling is likely to dictate the functional response and act as a developmental checkpoint for B-cell maturation.

Bcl-(xL) antagonism of BCR-coupled mitochondrial phospholipase A(2) signaling correlates with protection from apoptosis in WEHI-231 B cells.

Crosslinking of the antigen receptors on the immature B-cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. Commitment to such B-cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A2 activation, disruption of mitochondrial function, and cathepsin B activation. CD40 signaling has been reported to rescue WEHI-231 B cells from BCR-driven apoptosis primarily via up-regulation of the antiapoptotic protein Bcl-xL. Coupling of the BCR to the mitochondrial phospholipase A2-dependent apoptotic pathway can be prevented by rescue signals via CD40. We now show that overexpression of Bcl-xL can prevent mitochondrial phospholipase A2 activation, disruption of mitochondrial potential, and postmitochondrial execution of BCR-mediated apoptosis via cathepsin B activation. Moreover, overexpression of Bcl-xL protects WEHI-231 B cells from mitochondrial disruption and apoptosis resulting from culture with exogenous arachidonic acid, the product of phospholipase A2 action, suggesting that Bcl-xL may act to antagonize arachidonic acid-mediated disruption of mitochondrial integrity. However, although Bcl-xL expression can mimic CD40-mediated rescue of BCR-driven apoptosis, it cannot substitute for CD40 signaling in the reversal of BCR-mediated growth arrest of WEHI-231 B cells. Rather, CD40 signaling additionally induces conversion of arachidonic acid to prostaglandin E2 (PGE2), which promotes WEHI-231 B-cell proliferation by restoring the sustained, cycling extracellular signal-regulated/mitogen-activated protein kinase (ErkMAPkinase) signaling required for cell cycle progression.