Epithelial TGFß engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features.

The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.

The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult mouse gut.

The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. The signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. We set out to systematically compare the effect of two ECM classes, the interstitial matrix and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all cell types present in both cultures, albeit at different ratios. The collagen cultures contained a subset of cells enriched in fetal-like markers. In contrast, laminin increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture resembled gut development in vivo. The dramatic ECM remodelling was accompanied by a local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. Importantly, deletion of laminin in the adult mouse resulted in a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.

Loss of Cxcr2 in Myeloid Cells Promotes Tumour Progression and T Cell Infiltration in Invasive Bladder Cancer.

BACKGROUND: CXCR2 is a chemokine receptor expressed in myeloid cells, including neutrophils and macrophages. Pharmacological inhibition of CXCR2 has been shown to sensitize tumours to immune checkpoint inhibitor immunotherapies in some cancer types. OBJECTIVE: To investigate the effects of CXCR2 loss in regulation of tumour-infiltrating myeloid cells and their relationship to lymphocytes during bladder tumorigenesis. METHODS: Urothelial pathogenesis and immune contexture was investigated in an OH-BBN model of invasive bladder cancer with Cxcr2 deleted in myeloid cells (LysMCre Cxcr2 flox/flox ). CXCR2 gene alterations and expression in human muscle invasive bladder cancer were analysed in The Cancer Genome Atlas. RESULTS: Urothelial tumour pathogenesis was significantly increased upon Cxcr2 deletion compared to wildtype mice. This was associated with a suppression of myeloid cell infiltration in Cxcr2-deleted bladders shortly after the carcinogen induction. Interestingly, following a transient increase of macrophages at the outset of tumour formation, an increase in T cell infiltration was observed in Cxcr2-deleted tumours. The increased tumour burden in the Cxcr2-deleted bladder was largely independent of T cells and the status of immune suppression. The Cxcr2-deleted mouse model reflected the low CXCR2 mRNA range in human bladder cancer, which showed poor overall survival. CONCLUSIONS: In contrast to previous reports of increased CXCR2 signalling associated with disease progression and poor prognosis, CXCR2 was protective against bladder cancer during tumour initiation. This is likely due to a suppression of acute inflammation. The strategy for sensitizing checkpoint immunotherapy by CXCR2 inhibition in bladder cancer may benefit from an examination of immune suppressive status.

Oncogenic BRAF, unrestrained by TGFß-receptor signalling, drives right-sided colonic tumorigenesis.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFß signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFß-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFß-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

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.

MNK Inhibition Sensitizes KRAS-Mutant Colorectal Cancer to mTORC1 Inhibition by Reducing eIF4E Phosphorylation and c-MYC Expression.

KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995.

RUNX1 Is a Driver of Renal Cell Carcinoma Correlating with Clinical Outcome.

The recurring association of specific genetic lesions with particular types of cancer is a fascinating and largely unexplained area of cancer biology. This is particularly true of clear cell renal cell carcinoma (ccRCC) where, although key mutations such as loss of VHL is an almost ubiquitous finding, there remains a conspicuous lack of targetable genetic drivers. In this study, we have identified a previously unknown protumorigenic role for the RUNX genes in this disease setting. Analysis of patient tumor biopsies together with loss-of-function studies in preclinical models established the importance of RUNX1 and RUNX2 in ccRCC. Patients with high RUNX1 (and RUNX2) expression exhibited significantly poorer clinical survival compared with patients with low expression. This was functionally relevant, as deletion of RUNX1 in ccRCC cell lines reduced tumor cell growth and viability in vitro and in vivo. Transcriptional profiling of RUNX1-CRISPR-deleted cells revealed a gene signature dominated by extracellular matrix remodeling, notably affecting STMN3, SERPINH1, and EPHRIN signaling. Finally, RUNX1 deletion in a genetic mouse model of kidney cancer improved overall survival and reduced tumor cell proliferation. In summary, these data attest to the validity of targeting a RUNX1-transcriptional program in ccRCC. SIGNIFICANCE: These data reveal a novel unexplored oncogenic role for RUNX genes in kidney cancer and indicate that targeting the effects of RUNX transcriptional activity could be relevant for clinical intervention in ccRCC.

Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis.

The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in KrasG12D-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) ß-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically.

Author Correction: Loss of BCL9/9l suppresses Wnt driven tumourigenesis in models that recapitulate human cancer.

The original version of this Article contained an error in the spelling of the author Miryam Müller, which was incorrectly given as Miryam Müeller. This has now been corrected in both the PDF and HTML versions of the Article.

Loss of BCL9/9l suppresses Wnt driven tumourigenesis in models that recapitulate human cancer.

Different thresholds of Wnt signalling are thought to drive stem cell maintenance, regeneration, differentiation and cancer. However, the principle that oncogenic Wnt signalling could be specifically targeted remains controversial. Here we examine the requirement of BCL9/9l, constituents of the Wnt-enhanceosome, for intestinal transformation following loss of the tumour suppressor APC. Although required for Lgr5+ intestinal stem cells and regeneration, Bcl9/9l deletion has no impact upon normal intestinal homeostasis. Loss of BCL9/9l suppressed many features of acute APC loss and subsequent Wnt pathway deregulation in vivo. This resulted in a level of Wnt pathway activation that favoured tumour initiation in the proximal small intestine (SI) and blocked tumour growth in the colon. Furthermore, Bcl9/9l deletion completely abrogated ß-catenin driven intestinal and hepatocellular transformation. We speculate these results support the just-right hypothesis of Wnt-driven tumour formation. Importantly, loss of BCL9/9l is particularly effective at blocking colonic tumourigenesis and mutations that most resemble those that occur in human cancer.

Neutrophils: Homing in on the myeloid mechanisms of metastasis.

The metastasis cascade is complex and comprises several stages including local invasion into surrounding tissue, intravasation and survival of tumour cells in the circulation, and extravasation and colonisation of a distant site. It is increasingly clear that these processes are driven not only by signals within the tumour cells, but are also profoundly influenced by stromal cells and signals in the tumour microenvironment. Amongst the many cell types within the tumour microenvironment, immune cells such as lymphocytes, macrophages and neutrophils play a prominent role in tumour development and progression. Neutrophils, however, have only recently emerged as important players, particularly in metastasis. Here we review the current evidence suggesting a multi-faceted role for neutrophils in the metastatic cascade.

Proof of concept and feasibility studies examining the influence of combination ribose, adenine and allopurinol treatment on stroke outcome in the rat.

BACKGROUND: Cerebral ischaemia results in a rapid and profound depletion of adenosine triphosphate (ATP), the energy currency of the cell. This depletion leads to disruption of cellular homeostasis and cell death. Early replenishment of ATP levels might therefore have a neuroprotective effect in the injured brain. We have previously shown that the ATP precursors, D-ribose and adenine (RibAde), restored the reduced ATP levels in rat brain slices to values similar to those measured in the intact rodent brain. The aim of this study was to assess whether RibAde, either alone or in combination with the xanthine oxidase inhibitor allopurinol (RibAdeAll; to further increase the availability of ATP precursors), could improve outcome in an in vivo rodent model of transient cerebral ischaemia. METHODS: After 60 min occlusion of the middle cerebral artery, and upon reperfusion, rats were administered saline, RibAde, or RibAdeAll for 6 h. Baseline lesion volume was determined by diffusion-weighted MRI prior to reperfusion and final infarct volume determined by T2-weighted MRI at Day 7. Neurological function was assessed at Days 1, 3 and 7. RESULTS: Ischaemic lesion volume decreased between Days 1 and 7: a 50% reduction was observed for the RibAdeAll group, 38% for the RibAde group and 18% in the animals that received saline. Reductions in lesion size in treatment groups were accompanied by a trend for faster functional recovery. CONCLUSION: These data support the potential use of ribose, adenine and allopurinol in the treatment of cerebral ischaemic injury, especially since all compounds have been used in man.

Multiple congenital cardiovascular defects including type IV persistent truncus arteriosus in a Shetland pony - Short communication.

This case report describes the pathological findings of multiple congenital cardiac defects in a 2-year-old female Shetland pony with clinical signs of chronic respiratory distress. Persistent truncus arteriosus (PTA) type IV, interventricular septal defect, overriding aorta, pulmonary trunk agenesis, pulmonary arteries arising from the descending aorta, and compensatory right ventricular hypertrophy were observed.

CXCR2 Inhibition Profoundly Suppresses Metastases and Augments Immunotherapy in Pancreatic Ductal Adenocarcinoma.

CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.

Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

Oncogene-induced senescence (OIS) is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1-Cre Kras(G12D/+) Trp53(R172H/+) (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor-free survival in KPC mice with early-stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease.

CXCR2 inhibition suppresses acute and chronic pancreatic inflammation.

Pancreatitis is a significant clinical problem and the lack of effective therapeutic options means that treatment is often palliative rather than curative. A deeper understanding of the pathogenesis of both acute and chronic pancreatitis is necessary to develop new therapies. Pathological changes in pancreatitis are dependent on innate immune cell recruitment to the site of initial tissue damage, and on the coordination of downstream inflammatory pathways. The chemokine receptor CXCR2 drives neutrophil recruitment during inflammation, and to investigate its role in pancreatic inflammation, we induced acute and chronic pancreatitis in wild-type and Cxcr2(-/-) mice. Strikingly, Cxcr2(-/-) mice were strongly protected from tissue damage in models of acute pancreatitis, and this could be recapitulated by neutrophil depletion or by the specific deletion of Cxcr2 from myeloid cells. The pancreata of Cxcr2(-/-) mice were also substantially protected from damage during chronic pancreatitis. Neutrophil depletion was less effective in this model, suggesting that CXCR2 on non-neutrophils contributes to the development of chronic pancreatitis. Importantly, pharmacological inhibition of CXCR2 in wild-type mice replicated the protection seen in Cxcr2(-/-) mice in acute and chronic models of pancreatitis. Moreover, acute pancreatic inflammation was reversible by inhibition of CXCR2. Thus, CXCR2 is critically involved in the development of acute and chronic pancreatitis in mice, and its inhibition or loss protects against pancreatic damage. CXCR2 may therefore be a viable therapeutic target in the treatment of pancreatitis.

Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.