A framework for advancing our understanding of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.

Global copy number profiling of cancer genomes.

UNLABELLED: In this article, we introduce a robust and efficient strategy for deriving global and allele-specific copy number alternations (CNA) from cancer whole exome sequencing data based on Log R ratios and B-allele frequencies. Applying the approach to the analysis of over 200 skin cancer samples, we demonstrate its utility for discovering distinct CNA events and for deriving ancillary information such as tumor purity. AVAILABILITY AND IMPLEMENTATION: https://github.com/xfwang/CLOSE CONTACT: xuefeng.wang@stonybrook.edu or michael.krauthammer@yale.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.

Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.

A trimming-and-retrieving alignment scheme for reduced representation bisulfite sequencing.

UNLABELLED: Currently available bisulfite sequencing tools frequently suffer from low mapping rates and low methylation calls, especially for data generated from the Illumina sequencer, NextSeq. Here, we introduce a sequential trimming-and-retrieving alignment approach for investigating DNA methylation patterns, which significantly improves the number of mapped reads and covered CpG sites. The method is implemented in an automated analysis toolkit for processing bisulfite sequencing reads. AVAILABILITY AND IMPLEMENTATION: http://mysbfiles.stonybrook.edu/~xuefenwang/software.html and https://github.com/xfwang/BStools.

Oncogenic Ras induces inflammatory cytokine production by upregulating the squamous cell carcinoma antigens SerpinB3/B4.

Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inflammatory cytokine production, although the underlying mechanism remains unclear. Here we show that squamous cell carcinoma antigens 1 and 2 (SCCA1/2), members of the Serpin family of serine/cysteine protease inhibitors, are transcriptionally upregulated by oncogenic Ras via MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to inhibition of protein turnover, unfolded protein response, activation of NF-κB and is essential for Ras-mediated cytokine production and tumour growth. Analysis of human colorectal and pancreatic tumour samples reveals a positive correlation between Ras mutation, enhanced SCCA expression and IL-6 expression. These results indicate that SCCA is a Ras-responsive factor that plays an important role in Ras-associated cytokine production and tumorigenesis.

System-wide analysis reveals a complex network of tumor-fibroblast interactions involved in tumorigenicity.

Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted.

Two Distinct Categories of Focal Deletions in Cancer Genomes.

One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we've addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.

Pattern discovery and cancer gene identification in integrated cancer genomic data.

Large-scale integrated cancer genome characterization efforts including the cancer genome atlas and the cancer cell line encyclopedia have created unprecedented opportunities to study cancer biology in the context of knowing the entire catalog of genetic alterations. A clinically important challenge is to discover cancer subtypes and their molecular drivers in a comprehensive genetic context. Curtis et al. [Nature (2012) 486(7403):346-352] has recently shown that integrative clustering of copy number and gene expression in 2,000 breast tumors reveals novel subgroups beyond the classic expression subtypes that show distinct clinical outcomes. To extend the scope of integrative analysis for the inclusion of somatic mutation data by massively parallel sequencing, we propose a framework for joint modeling of discrete and continuous variables that arise from integrated genomic, epigenomic, and transcriptomic profiling. The core idea is motivated by the hypothesis that diverse molecular phenotypes can be predicted by a set of orthogonal latent variables that represent distinct molecular drivers, and thus can reveal tumor subgroups of biological and clinical importance. Using the cancer cell line encyclopedia dataset, we demonstrate our method can accurately group cell lines by their cell-of-origin for several cancer types, and precisely pinpoint their known and potential cancer driver genes. Our integrative analysis also demonstrates the power for revealing subgroups that are not lineage-dependent, but consist of different cancer types driven by a common genetic alteration. Application of the cancer genome atlas colorectal cancer data reveals distinct integrated tumor subtypes, suggesting different genetic pathways in colon cancer progression.

DDX5 regulates DNA replication and is required for cell proliferation in a subset of breast cancer cells.

UNLABELLED: Understanding factors required for DNA replication will enrich our knowledge of this important process and potentially identify vulnerabilities that can be exploited in cancer therapy. We applied an assay that measures the stability of maintenance of an episomal plasmid in human tissue culture cells to screen for new DNA replication factors. We identify an important role for DDX5 in G(1)-S-phase progression where it directly regulates DNA replication factor expression by promoting the recruitment of RNA polymerase II to E2F-regulated gene promoters. We find that the DDX5 locus is frequently amplified in breast cancer and that breast cancer-derived cells with amplification of DDX5 are much more sensitive to its depletion than breast cancer cells and a breast epithelial cell line that lacks DDX5 amplification. Our results show a novel role for DDX5 in cancer cell proliferation and suggest DDX5 as a therapeutic target in breast cancer treatment. SIGNIFICANCE: DDX5 is required for cell proliferation by controlling the transcription of genes expressing DNA replication proteins in cancer cells in which the DDX5 locus is amplified, and this has uncovered a dependence on DDX5 for cell proliferation. Given the high frequency of DDX5 amplification in breast cancer, our results highlight DDX5 as a promising candidate for targeted therapy of breast tumors with DDX5 amplification, and indeed we show that DDX5 inhibition sensitizes a subset of breast cancer cells to trastuzumab.

From cancer genomes to oncogenic drivers, tumour dependencies and therapeutic targets.

The analysis of human cancer by genome sequencing and various types of arrays has proved that many tumours harbour hundreds of genes that are mutated or substantially altered by copy number changes. But how many of these changes are meaningful? And how can we exploit these massive data sets to yield new targets for cancer treatment? In this Opinion article, we describe emerging approaches that aim to determine which altered genes are actually contributing to cancer, as well as their potential as therapeutic targets.

Activation of multiple cancer pathways and tumor maintenance function of the 3q amplified oncogene FNDC3B.

FNDC3B was recently identified in an oncogenomic screen for amplified oncogenes in hepatocellular carcinoma. It is located at 3q26 and is amplified in over 20% of cancers, usually as part of a broad amplified region encompassing the entire 3q arm. Consistent with an oncogenic role in multiple cancer types, we show here that overexpression of FNDC3B is capable of malignantly transforming mammary and kidney epithelial cells in addition to hepatocytes. To explore how FNDC3B transforms cells, we determined the cellular localization of its gene product and the cancer pathways that it activates. We found that the FNDC3B oncoprotein localizes to the Golgi network, and that its correct localization is essential for its transforming function. We found that overexpression of FNDC3B induces the epithelial-to-mesenchymal transition (EMT) and activates several cancer pathways, including PI3-kinase/Akt, Rb1 and TGFß signaling. For TGFß signaling, we analyzed the point in the pathway at which FNDC3B operates and obtained evidence that it induces expression of all three TGFß ligands and also promotes TGFBR1 cell-surface localization. We found that RNAi-mediated knockdown of FNDC3B in cancer cells with 3q amplification suppressed their clonogenicity and tumorigenicity, but that the same RNAi knockdown had no effect on single-copy 3q cancer cells. These results indicate that FNDC3B is an important oncogenic driver gene of the 3q amplicon, adding to the growing list of oncogenic drivers within this commonly amplified region.