Claudio R. Santos

Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

BACKGROUND Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

SREBP Activity Is Regulated by mTORC1 and Contributes to Akt-Dependent Cell Growth

Summary Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). Here we show that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. We also show that silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of dSREBP in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. Our results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth.

Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing.

Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73–75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development.

Lipid metabolism in cancer

Lipids form a diverse group of water‐insoluble molecules that include triacylglycerides, phosphoglycerides, sterols and sphingolipids. They play several important roles at cellular and organismal levels. Fatty acids are the major building blocks for the synthesis of triacylglycerides, which are mainly used for energy storage. Phosphoglycerides, together with sterols and sphingolipids, represent the major structural components of biological membranes. Lipids can also have important roles in signalling, functioning as second messengers and as hormones. There is increasing evidence that cancer cells show specific alterations in different aspects of lipid metabolism. These alterations can affect the availability of structural lipids for the synthesis of membranes, the synthesis and degradation of lipids that contribute to energy homeostasis and the abundance of lipids with signalling functions. Changes in lipid metabolism can affect numerous cellular processes, including cell growth, proliferation, differentiation and motility. This review will examine some of the alterations in lipid metabolism that have been reported in cancer, at both cellular and organismal levels, and discuss how they contribute to different aspects of tumourigenesis.

Functional Metabolic Screen Identifies 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 as an Important Regulator of Prostate Cancer Cell Survival

UNLABELLED Alterations in metabolic activity contribute to the proliferation and survival of cancer cells. We investigated the effect of siRNA-mediated gene silencing of 222 metabolic enzymes, transporters, and regulators on the survival of 3 metastatic prostate cancer cell lines and a nonmalignant prostate epithelial cell line. This approach revealed significant complexity in the metabolic requirements of prostate cancer cells and identified several genes selectively required for their survival. Among these genes was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), an isoform of phosphofructokinase 2 (PFK2). We show that PFKFB4 is required to balance glycolytic activity and antioxidant production to maintain cellular redox balance in prostate cancer cells. Depletion of PFKFB4 inhibited tumor growth in a xenograft model, indicating that it is required under physiologic nutrient levels. PFKFB4 mRNA expression was also found to be greater in metastatic prostate cancer compared with primary tumors. Taken together, these results indicate that PFKFB4 is a potential target for the development of antineoplastic agents. SIGNIFICANCE Cancer cells undergo several changes in their metabolism that promote growth and survival. Using an unbiased functional screen, we found that the glycolytic enzyme PFKFB4 is essential for prostate cancer cell survival by maintaining the balance between the use of glucose for energy generation and the synthesis of antioxidants. Targeting PFKFB4 may therefore present new therapeutic opportunities.

A new player in the orchestra of cell growth: SREBP activity is regulated by mTORC1 and contributes to the regulation of cell and organ size

Cell growth requires co-ordinated regulation of processes that provide metabolites for the synthesis of macromolecules such as proteins and membrane lipids. In recent years, a lot of emphasis has been placed on the activation of protein synthesis by mTORC1 (mammalian target of rapamycin complex 1). The contribution of anabolic pathways other than protein synthesis has only been considered recently. In the present paper, we discuss recent findings regarding the contribution of transcriptional regulation of lipogenesis genes by the SREBP (sterol-regulatory-element-binding protein) transcription factor, a central regulator of expression of lipogenic genes, to the control of cell size in vitro and cell and organ size in vivo.

SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome‐wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta‐analysis of five ccRCC expression datasets. MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra‐tumoural heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs. MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse‐free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC. Copyright

Systematic Evaluation of the Prognostic Impact and Intratumour Heterogeneity of Clear Cell Renal Cell Carcinoma Biomarkers

Background Candidate biomarkers have been identified for clear cell renal cell carcinoma (ccRCC) patients, but most have not been validated. Objective To validate published ccRCC prognostic biomarkers in an independent patient cohort and to assess intratumour heterogeneity (ITH) of the most promising markers to guide biomarker optimisation. Design, setting, and participants Cancer-specific survival (CSS) for each of 28 identified genetic or transcriptomic biomarkers was assessed in 350 ccRCC patients. ITH was interrogated in a multiregion biopsy data set of 10 ccRCCs. Outcome measurements and statistical analysis Biomarker association with CSS was analysed by univariate and multivariate analyses. Results and limitations A total of 17 of 28 biomarkers (TP53 mutations; amplifications of chromosomes 8q, 12, 20q11.21q13.32, and 20 and deletions of 4p, 9p, 9p21.3p24.1, and 22q; low EDNRB and TSPAN7 expression and six gene expression signatures) were validated as predictors of poor CSS in univariate analysis. Tumour stage and the ccB expression signature were the only independent predictors in multivariate analysis. ITH of the ccB signature was identified in 8 of 10 tumours. Several genetic alterations that were significant in univariate analysis were enriched, and chromosomal instability indices were increased in samples expressing the ccB signature. The study may be underpowered to validate low-prevalence biomarkers. Conclusions The ccB signature was the only independent prognostic biomarker. Enrichment of multiple poor prognosis genetic alterations in ccB samples indicated that several events may be required to establish this aggressive phenotype, catalysed in some tumours by chromosomal instability. Multiregion assessment may improve the precision of this biomarker. Patient summary We evaluated the ability of published biomarkers to predict the survival of patients with clear cell kidney cancer in an independent patient cohort. Only one molecular test adds prognostic information to routine clinical assessments. This marker showed good and poor prognosis results within most individual cancers. Future biomarkers need to consider variation within tumours to improve accuracy.

Regulation of the SREBP transcription factors by mTORC1.

In recent years several reports have linked mTORC1 (mammalian target of rapamycin complex 1) to lipogenesis via the SREBPs (sterol-regulatory-element-binding proteins). SREBPs regulate the expression of genes encoding enzymes required for fatty acid and cholesterol biosynthesis. Lipid metabolism is perturbed in some diseases and SREBP target genes, such as FASN (fatty acid synthase), have been shown to be up-regulated in some cancers. We have previously shown that mTORC1 plays a role in SREBP activation and Akt/PKB (protein kinase B)-dependent de novo lipogenesis. Our findings suggest that mTORC1 plays a crucial role in the activation of SREBP and that the activation of lipid biosynthesis through the induction of SREBP could be part of a regulatory pathway that co-ordinates protein and lipid biosynthesis during cell growth. In the present paper, we discuss the increasing amount of data supporting the potential mechanisms of mTORC1-dependent activation of SREBP as well as the implications of this signalling pathway in cancer.