Faculty Bibliography

To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology.

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) has produced extensive mass spectrometry based proteomics data for selected breast, colon and ovarian tumors from The Cancer Genome Atlas (TCGA). We have incorporated the CPTAC proteomics data into the cBioPortal to support easy exploration and integrative analysis of these proteomic datasets in the context of the clinical and genomics data from the same tumors. cBioPortal is an open source platform for exploring, visualizing, and analyzing multi-dimensional cancer genomics and clinical data. The public instance of the cBioPortal (http://cbioportal.org/) hosts more than 200 cancer genomics studies including all of the data from TCGA. Its biologist-friendly interface provides many rich analysis features, including a graphical summary of gene-level data across multiple platforms, correlation analysis between genes or other data types, survival analysis, and per-patient data visualization. Here, we present the integration of the CPTAC mass spectrometry based proteomics data into the cBioPortal, consisting of 77 breast, 95 colorectal, and 174 ovarian tumors that already have been profiled by TCGA for mutations, copy number alterations, gene expression, and DNA methylation. As a result, the CPTAC data can now be easily explored and analyzed in the cBioPortal in the context of clinical and genomics data. By integrating CPTAC data into cBioPortal, limitations of TCGA proteomics array data can be overcome while also providing a user-friendly web interface, a web API and an R client to query the mass spectrometry data together with genomic, epigenomic, and clinical data.

Recent advances in the multi-omics characterization necessitate knowledge integration across different data types that go beyond individual biomarker discovery. In this study, we apply independent component analysis (ICA) to human breast cancer proteogenomics data to retrieve mechanistic information. We show that as an unsupervised feature extraction method, ICA was able to construct signatures with known biological relevance on both transcriptome and proteome levels. Moreover, proteome and transcriptome signatures can be associated by their respective correlation with patient clinical features, providing an integrated description of phenotype-related biological processes. Our results demonstrate that the application of ICA to proteogenomics data could lead to pathway-level knowledge discovery. Potential extension of this approach to other data and cancer types may contribute to pan-cancer integration of multi-omics information.

Aberrant phospho-signaling is a hallmark of cancer. We investigated kinase-substrate regulation of 33,239 phosphorylation sites (phosphosites) in 77 breast tumors and 24 breast cancer xenografts. Our search discovered 2,134 quantitatively-correlated kinase-phosphosite pairs, enriching for and extending experimental or binding-motif predictions. Among the 91 kinases with auto-phosphorylation, elevated EGFR, ERBB2, PRKG1, and WNK1 phosphosignaling were enriched in basal, HER2-E, Luminal A, and Luminal B breast cancers, respectively, revealing subtype-specific regulation. CDKs, MAPKs, and ataxia-telangiectasia proteins were dominant, master regulators of substrate-phosphorylation, whose activities are not captured by genomic evidence. We unveiled phospho-signaling and druggable targets from 113 kinase-substrate pairs and cascades downstream of kinases, including AKT1, BRAF and EGFR. We further identified kinase-substrate-pairs associated with clinical or immune signatures and experimentally validated activated phosphosites of ERBB2, EIF4EBP1, and EGFR. Overall, kinase-substrate regulation revealed by the largest unbiased global phosphorylation data to date connects driver events to their signaling effects.

Objective: To better understand uterine cancer using an integrated, high-throughput strategy that evaluates patterns of protein expression, post-translational modification (PTM), somatic mutations, copy number variation (CNV), and patterns of protein-coding and noncoding RNA expression in parallel.
Method(s): State-of-the-art platforms for mass spectrometry, next-generation sequencing, and bioinformatics were used to comprehensively profile carefully annotated specimens of endometrial cancer and matched adjacent and nonmatched normal tissue. Normals were histologically assessed for their relative composition of endometrium and myometrium. Microsatellite instability (MSI) was categorized as high or low based on whole exome sequencing and patterns of genomic methylation. Clinical demographics, including tumor histology and patient comorbid conditions, were verified across multiple source sites.
Result(s): Use of mass spectrometry consistently identified ~11,000 distinct proteins for each cancer (n = 101) and normal specimen (n = 40) in the discovery dataset (matched endometrium low, n = 25; nonmatched endometrium high normal cores, n = 9; and matched pure myometrium, n = 6). These data reveal unique patterns of proteomic biomarkers and neoantigen expression that differ significantly between cancer histotypes and normals. Cancer-associated protein expression is driven, at least partly, by distinct patterns of mutations and genomic CNV. Our data also reveal novel patterns of circular RNA associated with disruptions in protein expression that vary by uterine cancer histotype. Last, proteogenomic integration reveals previously unrecognized mechanisms potentially responsible for modulating immune responses in MSI-H uterine cancers. Ongoing work includes integrating patterns of microRNA expression, protein phosphorylation, and acetylation into our analyses.
Conclusion(s): Comprehensive proteogenomic analyses have revealed novel patterns of protein, PTM, and circular RNA expression associated with uterine cancer. These observations will be discussed, emphasizing the novel metabolic and immunologic susceptibilities we have identified.
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Lung cancer is the leading cause of cancer death in both men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize tumor proteome and phosphoproteome changes by longitudinal, prospective collection of tumor tissue from an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for over seven years while undergoing HER2-directed therapy in combination with chemotherapy. We employed "Super-SILAC" and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and eight distinct metastatic progressive lymph nodes collected during these seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung in comparison to the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. ERBB2/HER2 protein expression was higher in lung, consistent with a higher degree of ERBB2 amplification in lung compared to the lymph node metastatic sites. To further interrogate the mass spectrometry data, a patient-specific database was built by incorporating all the somatic and germline variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built to confirm variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays revealed two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, expressed in lung and lymph node metastatic sites, respectively. The CDK12-G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells increased chemotherapy sensitivity which was rescued by wild type, but not CDK12-G879V expression, consistent with the complete resolution of the lung metastatic sites in this patient.

Purpose: Many studies have been shown that obesity along with joint injury is one of the most common risk factor in the development of osteoarthritis (OA). In a previous study we described that intra-articular injections of liposomal preparations of adenosine completely prevent progression and reverse cartilage loss in post-traumatic OA. TGF-beta signaling plays dual and opposing roles in cartilage health and chondrocyte life depending on the signals activated downstream. Activation of downstream signaling pathways for TGF-beta leading to localization of phospho-SMAD2/3 associated with maintenance of cartilage. In contrast nuclear localization of phospho-SMAD1/5/8 results in chondrocyte hypertrophy. Here we report that intraarticular injections of liposomal adenosine and A2AR agonist reverses OA in a post-traumatic OA model in rat and in an obesity related mice model. We moreover explore the role of TGF-beta signaling in this phenomenon. Method(s): Obesity-induced OA model: C57Bl6 mice (5-6 for each group, 12 weeks old) were fed a 60% fat diet (HFF mice) for 3months, after which received intraarticular injections (10 mul) of empty liposomes (Lipo) or liposomes containing the A2AR agonist CGS21680 (Lipo-CGS) or Adenosine (Lipo-Ado) into the knee every 10 days for 4 injections. Post-traumatic OA (PTOA) was induced in Sprague Dawley rats following non-surgical rupture of anterior cruciate ligament (ACL). Four weeks later rats were injected in the knee with 100ul of saline, Lipo or Lipo-CGS every 10 days (6 injections). RNA was isolated from chondrocytes in knee cartilage of rats treated as described above (3 from each group X 3 replicates) and subjected to RNAseq analysis. TC28a2 human chondrocyte cell line was used for in vitro experiments. Result(s): Lipo-CGS and Lipo-Ado reversed OA in the obesity and post traumatic OA model. Mouse knees had an OARSI score of 5.17+/-1.84 before treatment. Treatment with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33+/-0.81 and 1.83+/-0.98, respectively, p<0.001 vs pre-treatment; figure 1). In the PTOA model the OARSI score significantly decreases after Lipo-CGS and Lipo-Ado treatment compare to the saline group (OARSI: 1.28+/-0.39; 1.42+/-0.69; 2.97+/-0.0.75 respectively; p<0.05). Analysis of the transcriptome suggests that the treatment of Lipo-CGS promotes the up-regulation of genes involved in proliferation process and downregulations of genes responsible of apoptosis, cartilage catabolism and chondrocyte hypertrophy (Figure 2). TGF-beta expression was increased in deep layers of cartilage in the Lipo-CGS-treated rats and there was notable nuclear localization of phospho-SMAD2/3 in these chondrocytes. In contrast, phospho-SMAD1/5/8 was expressed in the nuclei of chondrocytes in the saline and LIPO-treated rats but not in the LIPO-CGS treated rats. Identical changes were observed in the knees of obese mice. To determine whether the effect of A2AR stimulation on TGF-beta signaling was direct or indirect we studied the effect of CGS21680 on nuclear phospho-SMAD expression in TC28a2 cells and found that CGS21680 increased nuclear phospho-SMAD2/3 and reduced nuclear phospho-SMAD1/5/8, as detected by immunofluorescence. Conclusion(s): Administration of an A2AR agonist to established OA knees reverses OA in rats and mice and shifts TGF-beta signaling from ALK1/SMAD1/5/8 to ALK5/SMAD2/3 in OA chondrocytes after activation of A2AR in 2 OA animal models.

Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly identified and quantitatively compared sites of replication initiation and termination in untransformed human cells. We found that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thereby ensuring global co-directionality of transcription and replication, particularly at gene 5' ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally reorients replication relative to transcription around gene 3' ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.

Background/UNASSIGNED:Transposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA. Results/UNASSIGNED:Here we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants), as well as somatically-acquired L1 insertions that distinguish cancer genomes from a patient's constitutional make-up. Conclusions/UNASSIGNED:TIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes.