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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

Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of specific panels of mRNAs, and is defined in large part by the ribosomal protein (RP) content, amongst other things. However, it is currently unknown to what extent ribosomal composition is heterogeneous across tissues, which is compounded by a lack of tools available to study it. Here we present dripARF, a method for detecting differential RP incorporation into the ribosome using Ribosome Profiling (Ribo-seq) data. We combine the ‘waste’ rRNA fragment data generated in Ribo-seq with the known 3D structure of the human ribosome to predict differences in the composition of ribosomes in the material being studied. We have validated this approach using publicly available data, and have revealed a potential role for eS25/RPS25 in development. Our results indicate that ribosome heterogeneity can be detected in Ribo-seq data, providing a new method to study this phenomenon. Furthermore, with dripARF, previously published Ribo-seq data provides a wealth of new information, allowing the identification of RPs of interest in many disease and normal contexts. dripARF is available as part of the ARF R package and can be accessed through https://github.com/fallerlab/ARF.

Motivation Ribosome Profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of rRNA fragments. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, they may perform suboptimally in Ribo-seq. In order to overcome this, it is possible to use custom biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos. Results Here, we first show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a ‘one-size-fits-all’ approach may be inefficient. Therefore we developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We experimentally show that Ribo-ODDR designed oligos outperform commercially available kits and lead to a significant increase in rRNA depletion in Ribo-seq. Availability and implementation Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR. Supplementary information Supplementary data are available at Bioinformatics online.

Protein synthesis is one of the most essential processes in every kingdom of life, and its dysregulation is a known driving force in cancer development. Multiple signaling pathways converge on the translation initiation machinery, and this plays a crucial role in regulating differential gene expression. In colorectal cancer, dysregulation of initiation results in translational reprogramming, which promotes the selective translation of mRNAs required for many oncogenic processes. The majority of upstream mutations found in colorectal cancer, including alterations in the WNT, MAPK, and PI3K\AKT pathways, have been demonstrated to play a significant role in translational reprogramming. Many translation initiation factors are also known to be dysregulated, resulting in translational reprogramming during tumor initiation and/or maintenance. In this review, we outline the role of translational reprogramming that occurs during colorectal cancer development and progression and highlight some of the most critical factors affecting the etiology of this disease.

Background & Aims The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. Methods Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. Results We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. Conclusions Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning., Graphical abstract

Cell size varies between cell types but is tightly regulated by cell-intrinsic and extrinsic mechanisms. Cell-size control is important for cell function and changes in cell size are frequently observed in cancer cells. Here we uncover a non-canonical role of SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription regulation, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Although most of SETD2’s cellular functions have been linked to this activity, several non-histone substrates of SETD2 have recently been identified – some of which have been linked to novel functions of SETD2 beyond chromatin regulation. Using multiple, independent perturbation strategies we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that this function is dependent on the catalytic activity of SETD2 but independent of H3K36 methylation. Paradoxically, ectopic overexpression of a decoy SRI domain also increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/beta-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1 alpha, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1 alpha stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

Increased protein synthesis supports the rapid 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 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 colorectal cancer, as well as other cancers where the Rpl24Bst mutation has a tumour suppressive effect in mouse models.

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

KRAS-mutant colorectal cancers (CRC) 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 CRC. 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 KRAS(G12D) models to rapamycin via suppression of c-MYC. We show that 45% of CRCs 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 co-targeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population who may benefit from its clinical application.

Ribosome profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet, several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of ribosomal RNA (rRNA) fragments, which frequently make up more than 90% of sequencing reads if not depleted. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, such kits may perform suboptimally in Ribo-seq. There is therefore potential to significantly increase the information that can be gleaned from Ribo-seq experiments. Here we show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a “one-size-fits-all” approach may result in inefficient rRNA depletion. In order to overcome this, it is possible to use custom-designed biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos. We have developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We show that Ribo-ODDR designed oligos lead to a significant increase in rRNA depletion, and increased sequencing depth as a result, providing substantial information that would otherwise have been lost. Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR

Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc-deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation., Wnt ligands are essential for intestinal homoeostasis and stem cell maintenance. Here, the authors show that reduction in Wnt secretion reduces the number of intestinal stem cells; this results in rapid fixation of mutated stem cells and accelerated adenoma formation due to lack of cell competition.

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects., Glucocorticoids (GC) are reported to block cancer cell proliferation, but the mode of action is unclear. Here the authors show that glucocorticoid receptor activation induces cancer cell dormancy in lung cancer by regulating CDKN1C expression through a distal enhancer, and these dormant cells are addicted to IGF-1R signalling pathway.

The glucocorticoid receptor directly regulates thousands of genes across the human genome in a cell-type specific manner, governing various aspects of homeostasis. The influence of the glucocorticoid receptor is also seen in various pathologies, including cancer, where it has been linked to tumorigenesis, metastasis, apoptosis resistance, and therapy bypass. Nonetheless, the direct genetic and molecular underpinnings of glucocorticoid action in cancer remain elusive. Here, we dissected the glucocorticoid receptor signalling axis and uncovered the mechanism of glucocorticoid-mediated cancer cell dormancy. Upon glucocorticoid receptor activation cancer cells undergo quiescence, subserved by cell cycle arrest through CDKN1C and reprogramming of signalling orchestrated via FOXO1/IRS2. Strikingly, co-expression of these three genes, directly regulated by glucocorticoid-induced chromatin looping, correlates with a benign molecular phenotype across human cancers, whereas triple loss is associated with increased expression of proliferation/aggressiveness markers. Finally, we show that the glucocorticoid receptor signalling axis is inactivated by alterations of either the chromatin remodelling complex or TP53 in vitro and in vivo. Our results indicate that the activation of the glucocorticoid receptor leads to cancer cell dormancy, which has several implications in terms of glucocorticoid use in cancer therapy.

During late gestation, villi extend into the intestinal lumen to dramatically increase the surface area of the intestinal epithelium, preparing the gut for the neonatal diet. Incomplete development of the intestine is the most common gastrointestinal complication in neonates, but the causes are unclear. We provide evidence in mice that Yin Yang 1 (Yy1) is crucial for intestinal villus development. YY1 loss in the developing endoderm had no apparent consequences until late gestation, after which the intestine differentiated poorly and exhibited severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration might function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find that necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the still unappreciated role of metabolic regulation during organogenesis, and suggests that it might contribute to neonatal gastrointestinal disorders.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1’s role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of wnt/β-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with nuclear HIF-1α in intestinal adenomas, pointing towards a functional importance of hypoxia-independent, i.e. non-canonical HIF-1 stabilization that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1α and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

Levels of the actin bundling protein fascin correlate with invasion and metastasis and reveal prognostic value in many epithelial carcinomas. However, we know very little about the potential role of fascin in melanoma. The purpose of this study is to compare fascin expression in primary melanomas and melanoma metastasis. Fascin expression was examined through the immunohistochemistry of paraffin embedded tissue microarrays including 560 cores of primary tumour and metastasis. Fascin expression was significantly elevated in 48 metastases compared with 254 primary tumours (P=0.034). In 187 patients with primary melanomas, fascin was not correlated with survival (P=0.067), whereas low fascin was significantly correlated with the presence of ulceration (P=0.005). Our results indicate that fascin status does not correlate with progression in melanoma. Upregulated fascin expression was detected in melanoma metastases, but was not correlated to patient outcome.

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.

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.

Background: The homeobox containing transcription factor MSX2 is a key regulator of embryonic development and has been implicated to have a role in breast and pancreatic cancer. Methods: Using a selection of two- and three-dimensional in vitro assays and tissue microarrays (TMAs), the clinical and functional relevance of MSX2 in malignant melanoma was explored. A doxycyline-inducible over-expression system was applied to study the relevance of MSX2 in vitro. For TMA construction, tumour material from 218 melanoma patients was used. Results: Ectopic expression of MSX2 resulted in the induction of apoptosis and reduced the invasive capacity of melanoma cells in three-dimensional culture. MSX2 over-expression was shown to affect several signalling pathways associated with cell invasion and survival. Downregulation of N-Cadherin, induction of p21 and inhibition of both BCL2 and Survivin were observed. Cytoplasmic MSX2 expression was found to correlate significantly with increased recurrence-free survival (P=0.008). Nuclear expression of MSX2 did not result in significant survival correlations, suggesting that the beneficial effect of MSX2 may be independent of its DNA binding activity. Conclusions: MSX2 may be an important regulator of melanoma cell invasion and survival. Cytoplasmic expression of the protein was identified as biomarker for good prognosis in malignant melanoma patients.

DNA methylation plays a major role in cancer by silencing tumour suppressor genes. In melanoma, only a discrete number of methylated genes have been identified so far. After the treatment of melanoma cells with a DNA methyltransferase inhibitor and subsequent transcriptomic profiling, we had identified earlier a cohort of melanoma progression-associated genes regulated by methylation. Here, we identified which of these genes are directly methylated in melanoma cell lines and tissues. First, we examined 16 genes by bisulphite sequencing in the WM793 isogenic cell line model series. Five of these genes (CYBA, FABP5, MT1E, TSPY1 and TAC1) displayed increased methylation in several invasive cell lines compared with the parental WM793 cells, indicating their involvement in progression. Next, we analyzed several matched primary/metastatic tumours using methylation-specific PCR, which revealed that MT1E (one of the five genes assessed) was methylated in the largest proportion of tumours. Examination of a larger cohort of samples showed that 1 of 17 (6%) of the benign naevi, 16 of 43 (37%) primary tumours and 6 of 13 (46%) of the metastases displayed MT1E methylation. In addition, ectopic over-expression of MT1E mediated sensitization to cisplatin-induced apoptosis. Overall, these studies suggest that MT1E is a potential tumour suppressor gene, whose loss may promote resistance to apoptosis-inducing therapies.

In this study, we used array-comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to examine genetic aberrations in melanoma cell lines and tissues. Array-comparative genomic hybridization revealed that the most frequent genetic changes found in melanoma cell lines were amplifications on chromosomes 7p and 20q, along with disruptions on Chr 9, 10, 11, 12, 22 and Y. Validation of the results using FISH on tissue microarrays (TMAs) identified TOP1 as being amplified in melanoma tissues. TOP1 amplification was detected in a high percentage (33%) of tumours and was associated with thicker, aggressive tumours. These results show that TOP1 amplification is associated with advanced tumours and poor prognosis in melanoma. These observations open the possibility that TOP1-targeted therapeutics may be of benefit in a particular subgroup of advanced stage melanoma patients.

Background and Aims:\ud 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. \ud \ud Methods:\ud We used KRasG12D p53R172H Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes.\ud \ud Results:\ud Pancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRasG12D p53R172H Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial−mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence.\ud \ud Conclusions:The actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.

Melanoma cells break down lysophosphatidic acid from the environment, creating a chemotactic gradient that the tumor cells then follow; this provides an explanation for the rapid metastasis of melanoma., The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient., Author Summary Melanoma is feared because it spreads very rapidly when tumours are relatively small. It is not known why this metastasis is so efficient and aggressive. In particular, it is not known what drives melanoma cells to start to migrate out from the tumour. Here, we have studied the chemical signals that guide the migration of melanoma cells. We find that a component of serum, lysophosphatidic acid (LPA), functions as a remarkably strong attractant for all of the melanoma cells that we examined. We also observe that melanoma cells rapidly break down LPA. We conclude that melanomas create their own gradients of LPA, with low LPA in the tumour and high LPA outside. Since melanoma cells are attracted by LPA, this LPA gradient around the melanomas serves as a signal that drives the tumour cells out into the surrounding skin and blood vessels. Finally, we show that such gradients exist in a mouse model of melanoma. Self-generated LPA gradients are therefore an intriguing new driver for melanoma dispersal.

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

DNA methylation plays a major role in cancer by silencing tumour suppressor genes. In melanoma, only a discrete number of methylated genes have been identified so far. After the treatment of melanoma cells with a DNA methyltransferase inhibitor and subsequent transcriptomic profiling, we had identified earlier a cohort of melanoma progression-associated genes regulated by methylation. Here, we identified which of these genes are directly methylated in melanoma cell lines and tissues. First, we examined 16 genes by bisulphite sequencing in the WM793 isogenic cell line model series. Five of these genes (CYBA, FABP5, MT1E, TSPY1 and TAC1) displayed increased methylation in several invasive cell lines compared with the parental WM793 cells, indicating their involvement in progression. Next, we analyzed several matched primary/metastatic tumours using methylation-specific PCR, which revealed that MT1E (one of the five genes assessed) was methylated in the largest proportion of tumours. Examination of a larger cohort of samples showed that 1 of 17 (6%) of the benign naevi, 16 of 43 (37%) primary tumours and 6 of 13 (46%) of the metastases displayed MT1E methylation. In addition, ectopic over-expression of MT1E mediated sensitization to cisplatin-induced apoptosis. Overall, these studies suggest that MT1E is a potential tumour suppressor gene, whose loss may promote resistance to apoptosis-inducing therapies.

MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies ( n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.

Melanoma is an aggressive disease that spreads quickly and is resistant to most therapeutic agents. In an effort to provide insight into the molecular basis of melanoma progression, the expression of 94 genes in 20 metastatic melanomas using a high-throughput real-time quantitative RT-PCR assay was analysed.A TaqMan low density array (LDA) was designed containing probes/primers directed towards a cohort of genes previously found to be differentially expressed in an isogenic cell line model of melanoma progression. For each sample, cDNA was prepared and added to the quantitative assay. The resulting data were then analysed for correlations with clinical data.Clustering analysis divided the melanomas into two major subgroups based on gene expression patterns. When analysed individually, several genes were associated with overall survival, depth and type of the primary tumour.We have identified a selection of genes linked to melanoma progression and patient outcome.

Introduction The loss of Apc, causing Wnt-mediated epithelial proliferation, is an early event in colorectal cancer (CRC) development. This hyperproliferative state requires signalling though the mTOR pathway, with the current paradigm suggesting that upregulation of translation initiation via phosphorylation of 4EBP1 is crucial. This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 function, would be ineffective in limiting development and progression of intestinal adenomas. Methods The inducible in vivo mouse models Lgr5Cre ER and VillinCre ER were used to selectively flox genes from intestinal stem cells and crypts respectively. mTOR complex 1 signalling was inhibited in Apc fl/fl mice either by rapamycin treatment or co-floxing the mTORC1 essential component Raptor. Translational status was assessed by sucrose gradient ultracentrifugation of intestinal epithelial extract from these mice and 35 S methionine incorporation and harringtonine chase assays on organoid cultures. The role of downstream mTORC1 effectors was established by assessing the intestinal regeneration following IR irradiation of 4EBP1/2 DKO , S6K1/2 DKO , rpS6 mut and eEF2k -/- mice. Survival studies for Apc fl/fl mice treated with rapamycin were performed both prior to, and on development of, symptoms Results mTORC1 activity is absolutely required for the proliferation of Apc deficient, but not wild type, intestinal crypts. Surprisingly, although protein synthesis is increased in Apc fl/fl crypts, it is translation elongation and not initiation that is the rate limiting step. Mechanistically, the inhibition of eukaryotic elongation factor (eEF2) kinase, to increase eEF2 activity downstream of mTORC1 and S6K is required for Wnt-mediated proliferation after IR irradiation. Treatment of established Apc fl/fl adenomas with rapamycin (which inhibits the mTORC1-S6K-eEF2k-eEF2 axis) arrests tumour growth and prolongs life. Furthermore, rapamycin treatment of mice immediately following homozygous Apc loss prevents the onset of symptoms. Conclusion These data show that intestinal adenoma formation and growth requires an mTOR mediated increase in translation elongation. Treatment of patients at high risk of developing CRC, such as those with Familial Adenomatous Polyposis, with Rapalogs may therefore be of therapeutic value. Disclosure of Interest None Declared.

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.

Background: P-Rex1 is a Rac-specific Rho GTPase guanine nucleotide exchange factor that has recently been implicated in cancer for the first time. As Rac has a central role in control of cell motility, we assessed the role of P-Rex1 in melanoma, a cancer where metastasis is the major cause of death. This was the first evaluation of P-Rex1 in a genetically modified animal model of cancer. We also investigated the function of P-Rex1 in melanoblasts, as the molecular pathways that drive their developmental migration are believed to underpin the movement of metastatic melanoma cells. Methods: P-Rex1−/− mice were crossed to mice carrying the DCT-lacZ transgene, a melanoblast reporter line, and a Tyr::NrasQ61K/°; INK4a−/− metastatic melanoma mouse model. Allograft tail vein (TV) injections were performed to further assess the endogenous function of P-Rex1 in metastasis. Panels of human melanoma cell lines and tissue were investigated with microarray, tissue culture, and immunohistochemical techniques. Results: P-Rex1−/− mice have a melanoblast migration defect during development (p=0.01), evidenced by a white belly. They were resistant to metastasis when crossed to the Tyr::NrasQ61K/°; INK4a−/− murine model of melanoma (1/30 mice with metastases vs 13/30 in control cohort; p=0.001). When melanocytes derived from this model were TV-injected into immune-competent allografts, endogenous P-Rex1 was found to facilitate metastatic incidence, growth, and organ spread following the intravasation stage of the metastatic cascade (p=0.02). Mechanistically, this was associated with P-Rex1 driving invasion in a Rac-dependent manner. In humans, P-Rex1 was elevated in the great majority of melanoma cell lines, with mRNA levels above the median statistically associated with metastasic propensity (p=0.005). Tissue immunohistochemistry revealed an association between tumor progression and P-Rex1 expression. Conclusions: P-Rex1 is a key component of melanoma progression, invasion and metastatic signaling, supporting the value of pharmacological inhibition of P-Rex1 activation of Rac for treatment of metastatic or high risk primary melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B147.

TO THE EDITOR Given the recent discovery of RAC1-activating mutations in melanoma, and our finding that PIP3-dependent Rac-exchanger 1 (PREX1) is overexpressed and drives metastasis in this cancer, an important question is to establish whether the functions of P-Rex1 are mediated specifically by Rac alone (Lindsay et al., 2011; Berger et al., 2012). Here we describe a Rac1-independent in vivo role for P-Rex1 through identification and characterization of a mouse coat color phenotype. P-Rex1 is a guanine-nucleotide exchange factor (GEF) for Rac, whose primary cell function is induction of actin-mediated membrane ruffling and lamellipodia formation at the leading edge of cell migration (Welch et al., 2002; Hill et al., 2005; Barber et al., 2007). To investigate this question we decided to examine the role of Rac1 and P-Rex1 in melanoblast development. Previously, we reported a “white belly” phenotype of mice with Prex1 deletion (Lindsay et al., 2011). Impaired melanoblast migration was mostly responsible for this phenotype, with melanoblasts lacking at the most distal points of migration (belly and paws). Constitutive deletion of Rac1 is embryonically lethal, but a coat color defect of mice with melanocyte-specific RAC1 abrogation (Tyr::Cre Rac1fl/fl) has also been described; these mice have a larger belly spot on their ventral side, suggesting that alternative Rho-GTPases can be activated to enable melanoblast migration to the perimeter of the Tyr::Cre Racfl/fl white belly (Sugihara et al., 1998; Li et al., 2011). A role for Rac1 in proliferation was also observed, as there was a marked reduction of melanoblast numbers in this phenotype. In line with these previous studies, and because mice with melanocyte-specific RAC1 abrogation require euthanization shortly after birth because of neurological problems, we used the same embryonic melanoblast reporter models to assess the downstream effects of P-Rex1 in vivo (Mackenzie et al., 1997; Mort et al., 2010; Li et al., 2011). Melanocyte-specific reporter mouse strains employed were Tyr::Cre Z/EG, which drives green fluorescent protein expression in the melanoblast lineage, and DCT::β-galactosidase (otherwise referred to as DCT-lacZ). First, we hypothesized that, if the effects of P-Rex1 were mediated exclusively via Rac1, double mutant Tyr::Cre Rac1fl/fl; P-Rex1−/− mice would exhibit the same coat color phenotype as Tyr::Cre Racfl/fl mice alone. However, Tyr::Cre Racfl/fl; P-Rex1−/− mice display a dramatic alteration in coat color phenotype from Tyr::Cre Racfl/fl mice (n=7; Figure 1a). The ventral and dorsal coats of these mice are almost entirely white, with hypo-pigmented limbs and tail. Graying pigmented areas were only observed in the head coat. We concluded from this experiment that P-Rex1 and Rac1 together constitute fundamental signaling components of the mouse coat color phenotype, with minimal rescue of melanoblast development conferred by other GEFs or Rho-GTPases. It was also clear that P-Rex1 must be able to exert phenotypic effects other than via Rac1. Figure 1 P-Rex1 and Rac1 are fundamental components of a mouse coat color phenotype. (a) Ventral coats of P-Rex1−/−, Tyr::Cre Rac1fl/fl and P-Rex1−/−;Tyr::Cre Rac1fl/flmice. Final photomicrograph shows dorsal and head coat of Tyr::Cre ... To explore the Rac1-independent effects of P-Rex1 further, we crossed Tyr::Cre Racfl/fl; P-Rex1−/− mice with mice carrying the melanoblast reporter DCT-lacZ transgene (methods detailed in Lindsay et al., 2011). Relative to Tyr::Cre Racfl/flmice or P-Rex1−/− embryos alone, Tyr::Cre Racfl/fl; P-Rex1−/− embryos at E15.5 displayed a substantial reduction in melanoblast numbers across their entire body (Figure 1b and c). To assess whether the cause of this reduction in melanoblast numbers could be accounted for by decreased proliferation ± increased cell death, we next treated our previously described primary immortalized Tyr::CrER2 INK4a−/− Racfl/fl melanocyte cell line with short interfering RNA to P-Rex1 (methods detailed in Li et al., 2012). The use of this model system also allowed us to delete Rac1 function when these cells were treated with 4-hydroxytamoxifen (OHT). We first used western blotting to confirm that efficient P-Rex1 knockdown and OHT-induced Rac deletion were achieved (Figure 1d). There was no increase in cleaved caspase-3 evident in the absence of P-Rex1 and/or Rac, suggesting that the reduced cell numbers observed in Tyr::Cre Racfl/fl; P-Rex1−/− embryos were not accounted for by increased cell death (Figure 1d). Growth curves and anti-BrdU immunofluorescence of the same cell lines confirmed that there was a reduced proliferation in P-Rex1-depleted cells, both in the presence and absence of OHT (Figure 1e; Supplementary Figure S1a and b online). Taken together, these results suggest, in addition to our previously reported effects of Rac1 deletion on mouse coat color, that the loss of a Rac1-independent proliferative effect of P-Rex1 also contributes to the coat color phenotype observed in Tyr::Cre Racfl/fl; P-Rex1−/− mice (Li et al., 2011). In line with our previous characterization of the P-Rex1 knockout phenotype alone, we next decided to delineate whether the coat color phenotype of Tyr::Cre Racfl/fl; P-Rex1−/− mice could also be a consequence of reduced melanoblast migration (Lindsay et al., 2011). To assess this, mice with the Z/EG double reporter transgene were crossed with Tyr::Cre Racfl/fl; P-Rex1−/−mice, driving green fluorescent protein expression in the melanoblast lineage (methods detailed in Lindsay et al., 2011; Figure 2a). Live imaging of melanoblasts was performed using E15.5 embryo skin from each genotype (Figure 2b; Supplementary Movies S1–3 online). Consistent with our previous work, significant reductions in migration speed were observed between wild-type, P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts (Figure 2c and d). However, there was no significant difference in speed between Tyr::Cre Racfl/fl; P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts alone, suggesting that there was no change in migratory characteristics to account for the Tyr::Cre Racfl/fl; P-Rex1−/− phenotype (Figure 2c and d). These results were matched by similar differences between the same genotypes when Euclidean distance was measured (Figure 2e), as well as no observable change in cell morphology evident in Tyr::Cre Racfl/fl; P-Rex1−/− compared with Tyr::Cre Racfl/fl melanoblasts alone (Supplementary Figure S1c online; methods detailed in Helmy and Azim, 2012). Finally, no cell death was seen in our melanoblast time-lapse movies of any genotype, again suggesting that P-Rex1 contributes to coat color phenotype by promoting cell proliferation using a Rac1-independent mechanism (Li et al., 2011; Supplementary Movies S1–3 online; methods detailed in Lindsay et al., 2011). Figure 2 P-Rex1 has no additional effect on migration compared with Rac1 alone. All experiments show embryo skin explants at E15.5 (a) Combined Z-stack confocal images of Z/EG melanoblasts from wild-type (control), P-Rex1−/−, Tyr::Cre Rac1fl/fl ... To conclude, we have elucidated a proliferative role of P-Rex1 when Rac1 is deleted. As E15.5 migratory characteristics are not altered in the Tyr::Cre Racfl/fl; P-Rex1−/− double mutant embryos, this suggests that the role of P-Rex1 in migration is almost exclusively mediated via Rac1. Here we focused on E15.5 embryos, a useful time point to observe the late migratory effects observed with previously described Prex and Rac phenotypes (Li et al., 2011; Lindsay et al., 2011). With a greater number of embryos, we would have performed further embryo time-point analyses at E13.5 to ensure there was no earlier melanoblast migratory deficit that could contribute to this phenotype, although even this experiment could not completely exclude such a possibility. One potential Rho-GTPase, RhoG, is a likely candidate for P-Rex1 interaction: it is the most structurally similar Rho-GTPase to Rac and has been shown to cooperate with Rac for induction of cell transformation (Roux et al., 1997). Moreover, there are distinct regulatory and functional similarities between P-Rex1 and Vav proteins, which have been characterized as the predominant GEFs required for RhoG activation (Samson et al., 2010; Lawson et al., 2011). Further studies are underway to investigate the phenotypes of these and other potential Rho-GTPases in melanoblast migration and melanomagenesis. Given that there is now considerable effort to generate Rac1 inhibitors, our data would suggest functions for proteins upstream of Rac1 that may become further therapeutic targets in melanoma. All experiments were conducted and approved in accordance with institutional and UK guidelines, and all animal studies were performed in accordance with local regulatory guidelines.