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.

Increased protein synthesis supports the rapid cell proliferation associated with cancer. The Rpl24Bst mutant mouse reduces the expression of the ribosomal protein RPL24 and has been used to suppress translation and limit tumorigenesis in multiple mouse models of cancer. Here, we show that Rpl24Bst also suppresses tumorigenesis and proliferation in a model of colorectal cancer (CRC) with two common patient mutations, Apc and Kras. In contrast to previous reports, Rpl24Bst mutation has no effect on ribosomal subunit abundance but suppresses translation elongation through phosphorylation of eEF2, reducing protein synthesis by 40% in tumour cells. Ablating eEF2 phosphorylation in Rpl24Bst mutant mice by inactivating its kinase, eEF2K, completely restores the rates of elongation and protein synthesis. Furthermore, eEF2K activity is required for the Rpl24Bst mutant to suppress tumorigenesis. This work demonstrates that elevation of eEF2 phosphorylation is an effective means to suppress colorectal tumorigenesis with two driver mutations. This positions translation elongation as a therapeutic target in CRC, as well as in other cancers where the Rpl24Bst mutation has a tumour suppressive effect in mouse models.

Carnitine palmitoyltransferase 2 (CPT2) has been demonstrated to act as a tumor promotor or suppressor in different types of cancers. However, little is known about the effect of CPT2 on colorectal cancer (CRC). In the present study, we analyzed CPT2 expression in CRC tissues and cells. CPT2 was overexpressed in CRC cell lines (SW480 and RKO), and its effects and molecular mechanism on the proliferation, glycolysis, stemness, and oxaliplatin sensitivity were investigated. The xenograft experiment was used to confirm the influence of CPT2 on CRC tumorigenesis in vivo. We found that CPT2 expression was significantly downregulated in CRC patients, and its lower expression was associated with the poor prognosis, large tumor size, advanced TNM stage, and poor histological grade differentiation of patients. Upregulation of CPT2 significantly inhibited the proliferation, glycolytic metabolism, cancer stem cell properties, and oxaliplatin resistance in CRC cells. Also, the increase of CPT2 inhibited tumorigenesis, stemness and glycolysis, while enhanced oxaliplatin sensitivity in mouse models. Mechanistically, CPT2 functioned via suppressing the activation of Wnt/β-catenin pathway through repressing ROS production. In conclusion, our results demonstrated that CPT2 was decreased in CRC, and CPT2 downregulation could trigger stemness and oxaliplatin resistance in CRC via activating the ROS/Wnt/β-catenin-induced glycolytic metabolism. This study indicates that CPT2 is a potential therapeutic target for CRC.

Germline or somatic loss-of-function mutations of fumarate hydratase (FH) predispose patients to an aggressive form of renal cell carcinoma (RCC). Since other than tumor resection there is no effective therapy for metastatic FH-deficient RCC, an accurate method for early diagnosis is needed. Although MRI or CT scans are offered, they cannot differentiate FH-deficient tumors from other RCCs. Therefore, finding noninvasive plasma biomarkers suitable for rapid diagnosis, screening, and surveillance would improve clinical outcomes. Taking advantage of the robust metabolic rewiring that occurs in FH-deficient cells, we performed plasma metabolomics analysis and identified 2 tumor-derived metabolites, succinyl-adenosine and succinic-cysteine, as excellent plasma biomarkers for early diagnosis. These 2 molecules reliably reflected the FH mutation status and tumor mass. We further identified the enzymatic cooperativity by which these biomarkers are produced within the tumor microenvironment. Longitudinal monitoring of patients demonstrated that these circulating biomarkers can be used for reporting on treatment efficacy and identifying recurrent or metastatic tumors.

Strong immune responses in primary colorectal cancer correspond with better patient survival following surgery compared with tumors with predominantly stromal microenvironments. However, biomarkers to identify patients with colorectal cancer liver metastases (CRLM) with good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary colorectal cancer and CRLM and to interrogate the underlying functional biology that drives disease progression. Samples from patients undergoing synchronous resection of primary colorectal cancer and CRLM were evaluated in detail through histological assessment, panel genomic and bulk transcriptomic assessment, IHC, and GeoMx spatial transcriptomics (ST) analysis. High immune infiltration of metastases was associated with improved cancer-specific survival. Bulk transcriptomic analysis was confounded by stromal content, but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for type II IFN signaling and MHC-class II antigen presentation. In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T cells and neutrophils with enrichment of Notch and TGFβ signaling pathways at the metastatic tumor center. In summary, histological assessment can stratify outcomes in patients undergoing synchronous resection of CRLM, suggesting that it has potential as a prognostic biomarker. Furthermore, ST analysis has revealed significant intratumoral and interlesional heterogeneity and identified the underlying transcriptomic programs driving each phenotype.

Anti-hypertensive agents are one of the most frequently used drugs worldwide. However, no blood pressure-lowering strategy is superior to placebo with respect to survival in diabetic hypertensive patients. Previous findings show that Wnt co-receptors LDL receptor-related proteins 5 and 6 (LRP5/6) can directly bind to several G protein-coupled receptors (GPCRs). Because angiotensin II type 1 receptor (AT1R) is the most important GPCR in regulating hypertension, this study examines the possible mechanistic association between LRP5/6 and their binding protein Dickkopf-1 (DKK1) and activation of the AT1R and further hypothesizes that the LRP5/6-GPCR interaction may affect hypertension and potentiate cardiac impairment in the setting of diabetes.

The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.

Cholangiocarcinoma (CC) is typically diagnosed at an advanced stage and is refractory to surgical intervention and chemotherapy. Despite a global increase in the incidence of CC, little progress has been made toward the development of treatments for this cancer. Here we utilized human tissue; CC cell xenografts; a p53-deficient transgenic mouse model; and a non-transgenic, chemically induced rat model of CC that accurately reflects both the inflammatory and regenerative background associated with human CC pathology. Using these systems, we determined that the WNT pathway is highly activated in CCs and that inflammatory macrophages are required to establish this WNT-high state in vivo. Moreover, depletion of macrophages or inhibition of WNT signaling with one of two small molecule WNT inhibitors in mouse and rat CC models markedly reduced CC proliferation and increased apoptosis, resulting in tumor regression. Together, these results demonstrate that enhanced WNT signaling is a characteristic of CC and suggest that targeting WNT signaling pathways has potential as a therapeutic strategy for CC.

Although chemotherapy for prostate cancer (PCa) can improve patient survival, some tumours are chemo-resistant. Tumour molecular profiles may help identify the mechanisms of drug action and identify potential prognostic biomarkers. We performed in vivo transcriptome profiling of pre- and post-treatment prostatic biopsies from patients with advanced hormone-naive prostate cancer treated with docetaxel chemotherapy and androgen deprivation therapy (ADT) with an aim to identify the mechanisms of drug action and identify prognostic biomarkers.

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.

Dietary guidelines suggest limiting daily sodium intake to <2,300 mg for the general population, and <1,500 mg/d for those with certain cardiovascular risk factors. Despite these recommendations, few Americans are able to achieve this goal. Identifying challenges in meeting these guidelines is integral for successful compliance. This analysis examined patterns and amount of daily sodium intake among participants with metabolic syndrome enrolled in a one-year dietary intervention study.

Senescence has been implicated as an important mechanism of tumour suppression in a number of human malignancies, including colorectal cancer (CRC). However, we still have a relatively poor understanding of how the underlying mutations that occur in cancer cause senescence and its relevance in vivo. The Apc gene is mutated in approximately 80% of CRC as the initiating event, but rarely elsewhere. In this study we have examined the capacity of Apc loss to induce senescence in the intestinal epithelium compared to the renal epithelium. Within the renal epithelium, loss of Apc function led to an induction of senescence, however, bypassing senescence through combined Apc and p21 or Ink4A gene deletion rapidly initiated renal carcinoma. Within the intestinal epithelium, loss of Apc did not induce senescence. Moreover, combined Apc and p21 or Ink4A loss had no impact upon tumourigenesis. Taken together, these results show that Apc loss in vivo invokes a senescence program in a context-dependent fashion, and implies senescence may play a key barrier to tumourigenesis in the kidney. However, in CRC, escape from senescence is likely to only be a barrier in cancers initiated by other mutations.

Pancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice.

Androgen receptor (AR) signalling and the PI3K pathway mediate survival signals in prostate cancer, and have been shown to regulate each other by reciprocal negative feedback, such that inhibition of one activates the other. Understanding the reciprocal regulation of these pathways is important for disease management as tumour cells can adapt and survive when either single pathway is inhibited pharmacologically. We recently carried out genome-wide exon-specific profiling of prostate cancer cells to identify novel androgen-regulated transcriptional events. Here we interrogated this dataset for novel androgen-regulated genes associated with the PI3K pathway. We find that the PI3K regulatory subunits PIK3R1 (p85α) and PIK3R3 (p55γ) are direct targets of the AR which are rapidly repressed by androgens in LNCaP cells. Further characterisation revealed that the PIK3CA p110α catalytic subunit is also indirectly regulated by androgens at the protein level. We show that PIK3R1 mRNA is significantly under-expressed in prostate cancer (PCa) tissue, and provide data to suggest a context-dependent regulatory mechanism whereby repression of the p85α protein by the AR results in destabilisation of the PI3K p110α catalytic subunit and downstream PI3K pathway inhibition that functionally affects the properties of prostate cancer cells.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a key determinant of progression to metastasis in a number of human cancers. In accordance with this proposed role in cancer cell invasion and metastasis, we find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell line is sufficient to drive multiple migratory and invasive phenotypes. The invasive phenotype is greatly enhanced by the presence of a gradient of serum or platelet-derived growth factor, and is dependent upon the expression of functional PDGF receptor β. Consistently, the invasiveness of WM852 melanoma cells, which endogenously express P-Rex1 and PDGFRβ, is opposed by siRNA of either of these proteins. Furthermore, the current model of P-Rex1 activation is advanced through demonstration of P-Rex1 and PDGFRβ as components of the same macromolecular complex. These data suggest that P-Rex1 has an influence on physiological migratory processes, such as invasion of cancer cells, both through effects upon classical Rac1-driven motility and a novel association with RTK signalling complexes.

MicroRNAs (miRNAs), which are genomically encoded small RNAs, negatively regulate target gene expression at the post-transcriptional level. Our recent study indicated that microRNA-155 (miR-155) might be negatively correlated with blood pressure, and it has been suggested that miR-155-mediated target genes could be involved in the cardiovascular diseases. Bioinformatic analyses predict that angiotensin II type 1 receptor (AT(1)R) is a miR-155 target gene. The present study investigated the potential role of miR-155 in regulating AT(1)R expression and phenotypic differentiation in rat aortic adventitial fibroblasts (AFs). Luciferase assay demonstrated that miR-155 suppressed AT(1)R 3'-UTR reporter construct activity. miR-155 overexpression in AFs did not reduce target mRNA levels, but significantly reduced target protein expression. In addition, AFs transfected with pSUPER/miR-155 exhibited reduced Ang II-induced ERK1/2 activation. miR-155 overexpression in cells attenuated Ang II-induced α-smooth muscle actin (α-SMA, produces myofibroblast) expression, but did not transform growth factor beta-1 (TGF-β1). This study demonstrated that miR-155 could have an important role in regulating adventitial fibroblast differentiation and contribute to suppression of AT(1)R expression.

The Adenomatous Polyposis Coli (APC) gene is mutated in the majority of colorectal cancers (CRCs). Loss of APC leads to constitutively active WNT signaling, hyperproliferation, and tumorigenesis. Identification of pathways that facilitate tumorigenesis after APC loss is important for therapeutic development. Here, we show that RAC1 is a critical mediator of tumorigenesis after APC loss. We find that RAC1 is required for expansion of the LGR5 intestinal stem cell (ISC) signature, progenitor hyperproliferation, and transformation. Mechanistically, RAC1-driven ROS and NF-κB signaling mediate these processes. Together, these data highlight that ROS production and NF-κB activation triggered by RAC1 are critical events in CRC initiation.