Search

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production. ispartof: NATURE METABOLISM vol:4 issue:6 pages:693-+ ispartof: location:Germany status: published

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet., Dietary serine and glycine starvation has emerged as a potential therapy for cancer. Here, the authors show that inhibition of PHGDH, which mediates the first step in the serine synthesis pathway, improves the therapeutic efficacy of serine depletion diet in mouse xenograft models.

Damaged or superfluous cells are typically eliminated by apoptosis. Although apoptosis is a cell-autonomous process, apoptotic cells communicate with their environment in different ways. Here we describe a mechanism whereby cells under apoptotic stress can promote survival of neighbouring cells. We find that upon apoptotic stress, cells release the growth factor FGF2, leading to MEK-ERK-dependent transcriptional upregulation of pro-survival BCL-2 proteins in a non-cell autonomous manner. This transient upregulation of pro-survival BCL-2 proteins protects neighbouring cells from apoptosis. Accordingly, we find in certain cancer types a correlation between FGF-signalling, BCL-2 expression and worse prognosis. In vivo, upregulation of MCL-1 occurs in an FGF-dependent manner during skin repair, which regulates healing dynamics. Importantly, either co-treatment with FGF-receptor inhibitors or removal of apoptotic stress restores apoptotic sensitivity to cytotoxic therapy and delays wound healing. These data reveal a pathway by which cells under apoptotic stress can increase resistance to cell death in surrounding cells. Beyond mediating cytotoxic drug resistance, this process also provides a potential link between tissue damage and repair., Apoptosis is a cellular process that eliminates damaged or superfluous cells. Here the authors show that cells undergoing apoptotic stresss secrete the growth factor FGF2, which upregulates pro-survival BCL-2 proteins in neighbouring cells, thereby promoting their survival.

Current nutritional recommendations are focused on energy, fat, carbohydrate, protein and vitamins. Less attention has been paid to the nutritional demand of one-carbon units for nucleotide and methionine synthesis. Here, we investigated the impact of sodium formate supplementation as a nutritional intervention to increase the dietary intake of one-carbon units. A cohort of six female and six male mice received 125 mM of sodium formate in the drinking water for three months. A control group of another six female and six male mice was also followed up for the same period of time. Tail vein blood samples were collected once a month and profiled with a haematology analyser. At the end of the study, blood and tissues were collected for metabolomics analysis and immune cell profiling. Formate supplementation had no significant physiological effect on male mice, except for a small decrease in body weight. Formate supplementation had no significant effect on the immune cell counts during the intervention or at the end of the study in either gender. In female mice, however, the body weight and spleen wet weight were significantly increased by formate supplementation, while the blood plasma levels of amino acids were decreased. Formate supplementation also increased the frequency of bifidobacteria, a probiotic bacterium, in the stools of female mice. We conclude that formate supplementation induces physiological changes in a gender-specific manner.

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.

Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. Methods: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. Results: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. Conclusions: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.

Formate overflow coupled to mitochondrial oxidative metabolism\ has been observed in cancer cell lines, but whether that takes place in the tumor microenvironment is not known. Here we report the observation of serine catabolism to formate in normal murine tissues, with a relative rate correlating with serine levels and the tissue oxidative state. Yet, serine catabolism to formate is increased in the transformed tissue of in vivo models of intestinal adenomas and mammary carcinomas. The increased serine catabolism to formate is associated with increased serum formate levels. Finally, we show that inhibition of formate production by genetic interference reduces cancer cell invasion and this phenotype can be rescued by exogenous formate. We conclude that increased formate overflow is a hallmark of oxidative cancers and that high formate levels promote invasion via a yet unknown mechanism., Serine catabolism to formate supplies one-carbon units for biosynthesis. Here the authors show that formate production in murine cancers with high oxidative metabolism exceeds the biosynthetic demand and that high formate levels promotes invasion of cancer cells.

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15(INK4b) and p21(CIP1). Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo. Core support from the MRC and grants from MRCT, CRUK and the AICR financially supported the research in J.G's laboratory. J.G. is also supported by the EMBO Young Investigator Programme

SummaryThe 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.

Serine catabolism results in formate efflux that exceeds anabolic demands for purine synthesis., Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors.

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through BrafV600E or KrasG12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5+ve stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5+ve cells also results in cSCC development. These findings indicate that LGR5+ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis., Cutaneous squamous cell carcinomas is a growing problem but the driver genes causing this remain poorly defined. Here, the authors demonstrate that inactivating driver mutations in TGFBR1 and TGFBR2 occur in vemurafenib-induced and sporadic cutaneous squamous cell carcinomas.

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis: Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high, risk of developing colorectal cancer.

Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear β-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors β-catenin and c-Myc. β-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being β-catenin-dependent but c-Myc-independent in the liver.

Summary During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term “minority MOMP.” Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis., Graphical Abstract, Highlights • MOMP can occur in a minority of mitochondria • Minority MOMP triggers caspase activity but fails to kill cells • Minority MOMP-induced caspase activity causes DNA damage and genomic instability • Minority MOMP promotes cellular transformation and tumorigenesis, During apoptosis, mitochondrial outer membrane permeabilization (MOMP) is widespread, leading to rapid cell death. Here, Ichim et al. demonstrate that MOMP can also be engaged in a minority of mitochondria without killing the cell. Instead, minority MOMP triggers caspase-dependent DNA damage and genomic instability, thereby promoting transformation and tumorigenesis.

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach.

The p53 tumour suppressor inhibits tumour development via various mechanisms such as apoptosis, inhibition of proliferation or the activation of senescence. Recently, several studies have indicated a novel role of p53 in the regulation of energy metabolism. Previously we have discovered TIGAR, a p53 target gene that acts as a fructose-2,6-bisphosphatase. TIGAR therefore can redirect glucose from the glycolytic pathway to the pentose phosphate pathway (PPP), which promotes NADPH production to generate reduced glutathione for protecting against ROS, and also ribose 5 phosphate production for nucleotide synthesis. In order to understand the function of TIGAR in vivo, we generated TIGAR deficient mice. We have determined a critical role of TIGAR in rapidly proliferating tissue, either for repair after damage or during tumor development.

Inactivation of the Apc gene is recognized as the key early event in the development of sporadic colorectal cancer (CRC), where its loss leads to constitutive activation of β-catenin/T-cell factor 4 signaling and hence transcription of Wnt target genes such as c-Myc. Our and other previous studies have shown that although cyclin D1 is required for adenoma formation, it is not immediately upregulated following Apc loss within the intestine, suggesting that proliferation following acute Apc loss may be dependent on another D-type cyclin. In this study, we investigated the expression and functional relevance of cyclin D2 following Apc loss in the intestinal epithelium. Cyclin D2 is upregulated immediately following Apc loss, which corresponded with a significant increase in cyclin-dependent kinase 4 (CDK4) and hyperphosphorylated Rb levels. Deficiency of cyclin D2 resulted in a reduction in enterocyte proliferation and crypt size within Apc-deficient intestinal epithelium. Moreover, cyclin D2 dramatically reduced tumor growth and development in ApcMin/+ mice. Importantly, cyclin D2 knockout did not affect proliferation of normal enterocytes, and furthermore, CDK4/6 inhibition also suppressed the proliferation of adenomatous cells and not normal cells from ApcMin/+ mice. Taken together, these results indicate that cyclin D–CDK4/6 complexes are required for the efficient proliferation of cells with deregulated Wnt signaling, and inhibiting this complex may be an effective chemopreventative strategy in CRC. Cancer Res; 70(20); 8149–58. ©2010 AACR.

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/β-catenin pathway, we challenged the allele combinations by genetically restricting intracellular β-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/β-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/β-catenin signaling capacity similar to that in the germline of the Apcmin mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apcmin mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/β-catenin pathway that regulate both physiological and pathophysiological conditions., Author Summary Germline or somatic mutations in genes are the underlying cause of many human diseases, most notably cancer. Interestingly though, even in situations where every cell of every tissue of an organism carries the same mutation (as is the case for germline mutations), some tissues are more susceptible to the development of disease over time than others. For example, in familial adenomatous polyposis (FAP), affected persons carry different germline mutations in the APC gene and are prone to developing cancers of the colon and the rectum—and, less frequently, cancers in other tissues such as stomach, liver, and bones. Here we utilize a panel of mutant mice with truncating or hypomorphic mutations in the Apc gene, resulting in different levels of activation of the Wnt/β-catenin pathway. Our results reveal that different pathophysiological outcomes depend on different permissive signaling thresholds in embryonic, intestinal, and liver tissues. Importantly, we demonstrate that reducing Wnt pathway activation by 50% is enough to prevent the manifestation of embryonic abnormalities and disease in the adult mouse. This raises the possibility of developing therapeutic strategies that modulate the activation levels of this pathway rather than trying to “repair” the mutation in the gene itself.

Background Differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE) provides a tractable model system for studying molecular events during early and inaccessible stages of murine development. PE formation is accompanied by extensive changes in gene expression both in vivo and in culture. One of the most dramatic is the ~10-fold decrease in transcriptional output by RNA polymerase (pol) III. This has been attributed to changes in activity of TFIIIB, a factor that is necessary and sufficient to recruit pol III to promoters. The goal of this study was to identify molecular changes that can account for the low activity of TFIIIB following F9 cell differentiation. Results Three essential subunits of TFIIIB decrease in abundance as F9 cells differentiate; these are Brf1 and Bdp1, which are pol III-specific, and TBP, which is also used by pols I and II. The decreased levels of Brf1 and Bdp1 proteins can be explained by reduced expression of the corresponding mRNAs. However, this is not the case for TBP, which is regulated post-transcriptionally. In proliferating cells, pol III transcription is stimulated by the proto-oncogene product c-Myc and the mitogen-activated protein kinase Erk, both of which bind to TFIIIB. However, c-Myc levels fall during differentiation and Erk becomes inactive through dephosphorylation. The diminished abundance of TFIIIB is therefore likely to be compounded by changes to these positive regulators that are required for its full activity. In addition, PE cells have elevated levels of the retinoblastoma protein RB, which is known to bind and repress TFIIIB. Conclusion The low activity of TFIIIB in PE can be attributed to a combination of changes, any one of which could be sufficient to inhibit pol III transcription. Declining levels of essential TFIIIB subunits and of activators that are required for maximal TFIIIB activity are accompanied by an increase in a potent repressor of TFIIIB. These events provide fail-safe guarantees to ensure that pol III output is appropriate to the diminished metabolic requirements of terminally differentiated cells.