Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

DNA Deamination Is Required for Human APOBEC3A-Driven Hepatocellular Carcinoma In Vivo.

Although the APOBEC3 family of single-stranded DNA cytosine deaminases is well-known for its antiviral factors, these enzymes are rapidly gaining attention as prominent sources of mutation in cancer. APOBEC3's signature single-base substitutions, C-to-T and C-to-G in TCA and TCT motifs, are evident in over 70% of human malignancies and dominate the mutational landscape of numerous individual tumors. Recent murine studies have established cause-and-effect relationships, with both human APOBEC3A and APOBEC3B proving capable of promoting tumor formation in vivo. Here, we investigate the molecular mechanism of APOBEC3A-driven tumor development using the murine Fah liver complementation and regeneration system. First, we show that APOBEC3A alone is capable of driving tumor development (without Tp53 knockdown as utilized in prior studies). Second, we show that the catalytic glutamic acid residue of APOBEC3A (E72) is required for tumor formation. Third, we show that an APOBEC3A separation-of-function mutant with compromised DNA deamination activity and wildtype RNA-editing activity is defective in promoting tumor formation. Collectively, these results demonstrate that APOBEC3A is a "master driver" that fuels tumor formation through a DNA deamination-dependent mechanism.

Functional and Structural Insights into a Vif/PPP2R5 Complex Elucidated Using Patient HIV-1 Isolates and Computational Modeling.

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G2/M cell cycle arrest. Vif initiates G2/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-ß/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis.IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G2/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G2/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.

Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis.

BACKGROUND & AIMS: The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. Deregulation of receptor tyrosine kinases (RTKs) is frequent in HCC, yet little is known about the molecular events that cooperate with RTKs and whether these cooperative events play an active role at the root of liver tumorigenesis. METHODS: A forward genetic screen was performed using Sleeping Beauty transposon insertional mutagenesis to accelerate liver tumour formation in a genetic context in which subtly increased MET RTK levels predispose mice to tumorigenesis. Systematic sequencing of tumours identified common transposon insertion sites, thus uncovering putative RTK cooperators for liver cancer. Bioinformatic analyses were applied to transposon outcomes and human HCC datasets. In vitro and in vivo (through xenografts) functional screens were performed to assess the relevance of distinct cooperative modes to the tumorigenic properties conferred by RTKs. RESULTS: We identified 275 genes, most of which are altered in patients with HCC. Unexpectedly, these genes are not restricted to a small set of pathway/cellular processes, but cover a large spectrum of cellular functions, including signalling, metabolism, chromatin remodelling, mRNA degradation, proteasome, ubiquitination, cell cycle regulation, and chromatid segregation. We validated 15 tumour suppressor candidates, as shRNA-mediated targeting confers tumorigenicity to RTK-sensitized cells, but not to cells with basal RTK levels. This demonstrates that the context of enhanced RTK levels is essential for their action in tumour initiation. CONCLUSION: Our study identifies unanticipated genetic interactions underlying gene cooperativity with RTKs in HCC. Moreover, these results show how subtly increased levels of wild-type RTKs provide a tumour permissive cellular environment allowing a large spectrum of deregulated mechanisms to initiate liver cancer. LAY SUMMARY: Receptor tyrosine kinases (RTKs) are among signals frequently deregulated in patients with hepatocellular carcinoma and their deregulation confers essential biological properties to cancer cells. We have applied a genetic method to randomly mutate large numbers of genes in the context of a mouse model with increased RTK levels, predisposed to develop liver cancer. We identified mechanisms that accelerate tumour formation in cooperation with enhanced RTK levels. The wide array of cellular functions among these cooperators illustrates an extraordinary capability of RTKs to render the liver more vulnerable to additional alterations, by priming cells for tumour initiation.

RNA sequencing of Sleeping Beauty transposon-induced tumors detects transposon-RNA fusions in forward genetic cancer screens.

Forward genetic screens using Sleeping Beauty (SB)-mobilized T2/Onc transposons have been used to identify common insertion sites (CISs) associated with tumor formation. Recurrent sites of transposon insertion are commonly identified using ligation-mediated PCR (LM-PCR). Here, we use RNA sequencing (RNA-seq) data to directly identify transcriptional events mediated by T2/Onc. Surprisingly, the majority (∼80%) of LM-PCR identified junction fragments do not lead to observable changes in RNA transcripts. However, in CIS regions, direct transcriptional effects of transposon insertions are observed. We developed an automated method to systematically identify T2/Onc-genome RNA fusion sequences in RNA-seq data. RNA fusion-based CISs were identified corresponding to both DNA-based CISs (Cdkn2a, Mycl1, Nf2, Pten, Sema6d, and Rere) and additional regions strongly associated with cancer that were not observed by LM-PCR (Myc, Akt1, Pth, Csf1r, Fgfr2, Wisp1, Map3k5, and Map4k3). In addition to calculating recurrent CISs, we also present complementary methods to identify potential driver events via determination of strongly supported fusions and fusions with large transcript level changes in the absence of multitumor recurrence. These methods independently identify CIS regions and also point to cancer-associated genes like Braf. We anticipate RNA-seq analyses of tumors from forward genetic screens will become an efficient tool to identify causal events.

APOBEC3B is an enzymatic source of mutation in breast cancer.

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.

Guanine holes are prominent targets for mutation in cancer and inherited disease.

Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

The somatic autosomal mutation matrix in cancer genomes.

DNA damage in somatic cells originates from both environmental and endogenous sources, giving rise to mutations through multiple mechanisms. When these mutations affect the function of critical genes, cancer may ensue. Although identifying genomic subsets of mutated genes may inform therapeutic options, a systematic survey of tumor mutational spectra is required to improve our understanding of the underlying mechanisms of mutagenesis involved in cancer etiology. Recent studies have presented genome-wide sets of somatic mutations as a 96-element vector, a procedure that only captures the immediate neighbors of the mutated nucleotide. Herein, we present a 32 × 12 mutation matrix that captures the nucleotide pattern two nucleotides upstream and downstream of the mutation. A somatic autosomal mutation matrix (SAMM) was constructed from tumor-specific mutations derived from each of 909 individual cancer genomes harboring a total of 10,681,843 single-base substitutions. In addition, mechanistic template mutation matrices (MTMMs) representing oxidative DNA damage, ultraviolet-induced DNA damage, (5m)CpG deamination, and APOBEC-mediated cytosine mutation, are presented. MTMMs were mapped to the individual tumor SAMMs to determine the maximum contribution of each mutational mechanism to the overall mutation pattern. A Manhattan distance across all SAMM elements between any two tumor genomes was used to determine their relative distance. Employing this metric, 89.5% of all tumor genomes were found to have a nearest neighbor from the same tissue of origin. When a distance-dependent 6-nearest neighbor classifier was used, 10.4% of the SAMMs had an Undetermined tissue of origin, and 92.2% of the remaining SAMMs were assigned to the correct tissue of origin. [corrected]. Thus, although tumors from different tissues may have similar mutation patterns, their SAMMs often display signatures that are characteristic of specific tissues.

Non-B DB v2.0: a database of predicted non-B DNA-forming motifs and its associated tools.

The non-B DB, available at http://nonb.abcc.ncifcrf.gov, catalogs predicted non-B DNA-forming sequence motifs, including Z-DNA, G-quadruplex, A-phased repeats, inverted repeats, mirror repeats, direct repeats and their corresponding subsets: cruciforms, triplexes and slipped structures, in several genomes. Version 2.0 of the database revises and re-implements the motif discovery algorithms to better align with accepted definitions and thresholds for motifs, expands the non-B DNA-forming motifs coverage by including short tandem repeats and adds key visualization tools to compare motif locations relative to other genomic annotations. Non-B DB v2.0 extends the ability for comparative genomics by including re-annotation of the five organisms reported in non-B DB v1.0, human, chimpanzee, dog, macaque and mouse, and adds seven additional organisms: orangutan, rat, cow, pig, horse, platypus and Arabidopsis thaliana. Additionally, the non-B DB v2.0 provides an overall improved graphical user interface and faster query performance.

Distinct mechanisms of PTEN inactivation in dogs and humans highlight convergent molecular events that drive cell division in the pathogenesis of osteosarcoma.

A hallmark of osteosarcoma in both human and canine tumors is somatic fragmentation and rearrangement of chromosome structure which leads to recurrent increases and decreases in DNA copy number. The PTEN gene has been implicated as an important tumor suppressor in osteosarcoma via forward genetic screens. Here, we analyzed copy number changes, promoter methylation and transcriptomes to better understand the role of PTEN in canine and human osteosarcoma. Reduction in PTEN copy number was observed in 23 of 95 (25%) of the canine tumors examined leading to corresponding decreases in PTEN transcript levels from RNA-Seq samples. Unexpectedly, canine tumors with an intact PTEN locus had higher levels of PTEN transcripts than human tumors. This variation in transcript abundance was used to evaluate the role of PTEN in osteosarcoma biology. Decreased PTEN copy number and transcript level was observed in - and likely an important driver of - increases in cell cycle transcripts in four independent canine transcriptional datasets. In human osteosarcoma, homozygous copy number loss was not observed, instead increased methylation of the PTEN promoter was associated with increased cell cycle transcripts. Somatic modification of PTEN, either by homozygous deletion in dogs or by promoter methylation in humans, is clinically relevant to osteosarcoma, because the cell cycle related transcripts are associated with patient outcomes. The PTEN gene is part of a syntenic rearrangement unique to the canine genome, making it susceptible to somatic loss of both copies of distal chromosome 26 which also includes the FAS death receptor. SIGNIFICANCE STATEMENT: PTEN function is abrogated by different mechanisms in canine and human osteosarcoma tumors leading to uncontrolled cell cycling. Somatic loss of this canine specific syntenic region may help explain why the canine genome appears to be uniquely susceptible to osteosarcoma. Syntenic arrangement, in the context of copy number change, may lead to synergistic interactions that in turn modify species specific cancer risk. Comparative models of tumorigenesis may utilize different driver mechanisms.

APOBEC3B regulates R-loops and promotes transcription-associated mutagenesis in cancer.

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.

In Vitro Insertional Mutagenesis Screen Identifies Novel Genes Driving Breast Cancer Metastasis.

Metastasis, a complex, multistep process, is responsible for the overwhelming majority of cancer-related deaths. Despite its devastating consequences, it is not possible to effectively treat cancer that has spread to vital organs, the mechanisms leading to metastasis are still poorly understood, and the catalog of metastasis promoting genes is still incomprehensive. To identify new driver genes of metastasis development, we performed an in vitro Sleeping Beauty transposon-based forward genetic screen in nonmetastatic SKBR3 human breast cancer cells. Boyden chamber-based matrix invasion assays were used to harvest cells that acquired a de novo invasive phenotype. Using targeted RNA sequencing data from 18 pools of invasive cells, we carried out a gene-centric candidate gene prediction and identified established and novel metastasis driver genes. Analysis of these genes revealed their association with metastasis related processes and we further established their clinical relevance in metastatic breast cancer. Two novel candidate genes, G protein-coupled receptor kinase interacting ArfGAP 2 (GIT2) and muscle-associated receptor tyrosine kinase (MUSK), were functionally validated as metastasis driver genes in a series of in vitro and in vivo experimental metastasis models. We propose that our robust and scalable approach will be a useful addition to the toolkit of methodologic resources used to identify genes driving cancer metastasis. IMPLICATIONS: Novel metastasis drivers were identified in a human breast cancer cell line by performing an in vitro, Sleeping Beauty transposon-based forward genetic screen and an RNA fusion-based candidate gene prediction.

R-Spondins 2 and 3 Are Overexpressed in a Subset of Human Colon and Breast Cancers.

Wnt signaling is activated in many cancer types, yet targeting the canonical Wnt pathway has been challenging for cancer therapy. The pathway might be effectively targeted at many levels depending on the mechanism by which it has become hyperactive. Recently, mouse genetic screens have found that R-spondins (RSPOs) act as oncogenes. Evidence includes recurrent genomic rearrangements that led to increased RSPO2 or RSPO3 expression in human colorectal adenocarcinomas, exclusive of APC mutations. RSPOs modulate Wnt signaling to promote epithelial cell proliferation and survival. These secreted proteins modulate Wnt signaling by binding to G-coupled receptors LGR4/5/6, ultimately inhibiting frizzled membrane clearance by RNF43 and ZNRF3. They also exert their function independent of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) by binding to ZNRF3 and RNF43. This results in increased ß-catenin concentration that, after translocation to the nucleus, acts as a transcriptional coactivator of genes necessary for proliferation and cell survival. In this article, we aimed to identify the role of RSPOs in colon and breast cancers by using in silico and in vitro studies. We found that expression of RSPO2 and RSPO3 at high levels characterized a subset of colorectal cancers (CRCs). RSPO2 expression was found to characterize a subset of triple-negative breast cancers. In both instances, increased expression of RSPOs was associated with an activated Wnt signaling gene expression profile. Furthermore, knockdown of RSPO2 decreased Wnt signaling and proliferation in human breast cancer cells. Our findings show and confirm that RSPO2 and RSPO3 expression is upregulated in a subset of colorectal adenocarcinomas and breast cancers and that both are attractive druggable oncoprotein targets against such cancers. We also describe novel fusion transcripts that occur in CRC.

PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER+ breast cancer.

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.

Novel modulation factor quantifies the role of water molecules in protein interactions.

Water molecules decrease the potential of mean force of a hydrogen bond (H-bond), as well as modulate (de)solvation forces, but exactly how much has not been easy to determine. Crystallographic water molecules provide snapshots of optimal solutions for the role of solvent in protein interactions, information that is often ignored by implicit solvent models. Motivated by high-resolution crystal structures, we describe a simple quantitative approach to explicitly incorporate the role of molecular water in protein interactions. Applications to protein-DNA interactions show that the accuracy of binding free-energy estimates improves significantly if a distinction is made between H-bonds that are desolvated (or only contact crystal waters), solvated by mobile waters trapped at the binding interface, or partially solvated through connections to bulk water. These different environments are modeled by a unique "water" scaling factor that decreases or increases the strength of hydrogen bonds depending on whether water contacts the acceptor or donor atoms or the bond is fully desolvated, respectively. Our empirical energies are fully consistent with mobile water molecules having a strong polarization effect in direct intermolecular interactions.

APOBEC3A catalyzes mutation and drives carcinogenesis in vivo.

The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members, including APOBEC3B, fail to promote liver tumor formation. Tumor DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor datasets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.

R-spondin 2 Drives Liver Tumor Development in a Yes-Associated Protein-Dependent Manner.

Each year, more than 25,000 people succumb to liver cancer in the United States, and this neoplasm represents the second cause of cancer-related death globally. R-spondins (RSPOs) are secreted regulators of Wnt signaling that function in development and promote tissue stem cell renewal. In cancer, RSPOs 2 and 3 are oncogenes first identified by insertional mutagenesis screens in tumors induced by mouse mammary tumor virus and by transposon mutagenesis in the colonic epithelium of rodents. RSPO2 has been reported to be activated by chromosomal rearrangements in colorectal cancer and overexpressed in a subset of hepatocellular carcinoma. Using human liver tumor gene expression data, we first discovered that a subset of liver cancers were characterized by high levels of RSPO2 in contrast to low levels in adjacent nontumor tissue. To determine if RSPOs are capable of inducing liver tumors, we used an in vivo model from which we found that overexpression of RSPO2 in the liver promoted Wnt signaling, hepatomegaly, and enhanced liver tumor formation when combined with loss of transformation-related protein 53 (Trp53). Moreover, the Hippo/yes-associated protein (Yap) pathway has been implicated in many human cancers, influencing cell survival. Histologic and gene expression studies showed activation of Wnt/ß-catenin and Hippo/Yap pathways following RSPO2 overexpression. We demonstrate that knockdown of Yap1 leads to reduced tumor penetrance following RSPO2 overexpression in the context of loss of Trp53. Conclusion: RSPO2 overexpression leads to tumor formation in the mouse liver in a Hippo/Yap-dependent manner. Overall, our results suggest a role for Yap in the initiation and progression of liver tumors and uncover a novel pathway activated in RSPO2-induced malignancies. We show that RSPO2 promotes liver tumor formation in vivo and in vitro and that RSPO2's oncogenic activity requires Hippo/Yap activation in hepatocytes. Both RSPO2 and YAP1 are suggested to represent novel druggable targets in Wnt-driven tumors of the liver.

Insulin Receptor Substrate Suppression by the Tyrphostin NT157 Inhibits Responses to Insulin-Like Growth Factor-I and Insulin in Breast Cancer Cells.

Insulin and insulin-like growth factor (IGF) signaling systems regulate breast cancer growth, progression, and metastasis. The insulin receptor substrates 1 and 2 (IRS1/2) transduce signaling from the type I IGF receptor (IGF-IR) and insulin receptor (InR) to mediate the biological effects of receptor activation. In breast cancer, IRS-1 plays a critical role in cancer cell proliferation while IRS-2 is associated with motility and metastasis. NT157, a small-molecule tyrphostin, downregulates IRS proteins in several model systems. In breast cancer cells, NT157 treatment suppressed IRS protein expression in a dose-dependent manner. Exposure to NT157 inhibited the activation of downstream signaling mediated by the IRS proteins. NT157 induced a MAPK-dependent serine phosphorylation of IRS proteins which resulted in disassociation between IRS proteins and their receptors resulting in IRS degradation. In estrogen receptor-α-positive (ERα+) breast cancer cells (MCF-7 and T47D), NT157 also resulted in cytoplasmic ERα downregulation likely because of disruption of an IRS-1-IGF-IR/InR/ERα complex. NT157 decreased S phase fraction, monolayer, and anchorage-independent growth after IGF/insulin treatment in ERα+ breast cancer cells. NT157 downregulation of IRS protein expression also sensitized ERα+ breast cancer cells to rapamycin. Moreover, NT157 inhibited the growth of tamoxifen-resistant ERα+ breast cancer cells. Given that both IGF-IR and InR play a role in cancer biology, targeting of IRS adaptor proteins may be a more effective strategy to inhibit the function of these receptors.

Mutation Signatures Including APOBEC in Cancer Cell Lines.

BACKGROUND: Multiple endogenous and exogenous sources of DNA damage contribute to the overall mutation burden in cancer, with distinct and overlapping combinations contributing to each cancer type. Many mutation sources result in characteristic mutation signatures, which can be deduced from tumor genomic DNA sequences. Examples include spontaneous hydrolytic deamination of methyl-cytosine bases in CG motifs (AGEING signature) and C-to-T and C-to-G mutations in 5'-TC(A/T) motifs (APOBEC signature). METHODS: The deconstructSigs R package was used to analyze single base substitution mutation signatures in over 1000 cancer cell lines. Two additional approaches were used to analyze the APOBEC mutation signature. RESULTS: Most cell lines show evidence for multiple mutation signatures. For instance, the AGEING signature, which is the largest source of mutation in most primary tumors, predominates in the majority of cancer cell lines. The APOBEC mutation signature is enriched in cancer cell lines from breast, lung, head/neck, bladder, and cervical cancers, where this signature also comprises a large fraction of all mutations. CONCLUSIONS: The single base substitution mutation signatures of cancer cell lines often reflect those of the original tumors from which they are derived. Cancer cell lines with enrichments for distinct mutation signatures such as APOBEC have the potential to become model systems for fundamental research on the underlying mechanisms and for advancing clinical strategies to exploit these processes.