Mark A. Eckert

Genomics of Ovarian Cancer Progression Reveals Diverse Metastatic Trajectories Including Intraepithelial Metastasis to the Fallopian Tube

Accumulating evidence has supported the fallopian tube rather than the ovary as the origin for high-grade serous ovarian cancer (HGSOC). To understand the relationship between putative precursor lesions and metastatic tumors, we performed whole-exome sequencing on specimens from eight HGSOC patient progression series consisting of serous tubal intraepithelial carcinomas (STIC), invasive fallopian tube lesions, invasive ovarian lesions, and omental metastases. Integration of copy number and somatic mutations revealed patient-specific patterns with similar mutational signatures and copy-number variation profiles across all anatomic sites, suggesting that genomic instability is an early event in HGSOC. Phylogenetic analyses supported STIC as precursor lesions in half of our patient cohort, but also identified STIC as metastases in 2 patients. Ex vivo assays revealed that HGSOC spheroids can implant in the fallopian tube epithelium and mimic STIC lesions. That STIC may represent metastases calls into question the assumption that STIC are always indicative of primary fallopian tube cancers. SIGNIFICANCE We find that the putative precursor lesions for HGSOC, STIC, possess most of the genomic aberrations present in advanced cancers. In addition, a proportion of STIC represent intraepithelial metastases to the fallopian tube rather than the origin of HGSOC. Cancer Discov; 6(12); 1342-51. ©2016 AACR.See related commentary by Swisher et al., p. 1309This article is highlighted in the In This Issue feature, p. 1293.

Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium.

Who are the long-term survivors of high grade serous ovarian cancer?

Although the median survival for epithelial ovarian cancer (EOC) is <5years, approximately 15% of patients will survive for >10years. A better understanding of these exceptional responders could reveal opportunities to improve the dismal prognosis of most EOC patients. In this review, we examine the clinical and genomic features that have been associated with long-term survival, which is generally defined as survival of >7-10years after initial diagnosis. Clinical features influencing long-term survival have been best reported in large retrospective population-based studies. These studies find that long-term survival is associated with previously validated prognostic factors, including younger age at diagnosis, earlier clinicopathologic stage, lower grade, non-serous histology, absence of ascites, primary debulking surgery, and optimal cytoreduction at primary surgery. Duration of survival after a recurrence also contributes to long-term survival and depends both on recurrence location and response to subsequent chemotherapy or surgery. Germline BRCA mutations, although associated with short-term chemosensitivity, do not appear to improve long-term survival. Unfortunately, the relative lack of recurrent somatic mutations in EOC has made the identification of genomic signatures associated with long-term survival difficult. Although six independent gene expression analyses of long-term survivors (LTS) have identified signatures associated with prolonged survival, different gene sets are identified in each study. Genes differentially expressed in tumors of LTS are broadly involved in cell proliferation, tumor-stromal interactions, the cytoskeleton, metabolism of nutrients, and immune/stress response. We anticipate that consistent selection of control and LTS groups, combined with the use of emerging transcriptomic, epigenomic, and proteomic platforms, is likely to identify conserved features associated with long-term survival. Further elucidating the factors contributing to long-term survival has the potential to contribute to our understanding of the biology of ovarian cancer, with the goal of improving the survival of all EOC patients.

Loss of BRCA1 in the cells of origin of ovarian cancer induces glycolysis: A window of opportunity for ovarian cancer chemoprevention

Mutations in the breast cancer susceptibility gene 1 (BRCA1) are associated with an increased risk of developing epithelial ovarian cancer. However, beyond the role of BRCA1 in DNA repair, little is known about other mechanisms by which BRCA1 impairment promotes carcinogenesis. Given that altered metabolism is now recognized as important in the initiation and progression of cancer, we asked whether the loss of BRCA1 changes metabolism in the cells of origin of ovarian cancer. The findings show that silencing BRCA1 in ovarian surface epithelial and fallopian tube cells increased glycolysis. Furthermore, when these cells were transfected with plasmids carrying deleterious BRCA1 mutations (5382insC or the P1749R), there was an increase in hexokinase-2 (HK2), a key glycolytic enzyme. This effect was mediated by MYC and the STAT3. To target the metabolic phenotype induced by loss of BRCA1, a drug-repurposing approach was used and aspirin was identified as an agent that counteracted the increase in HK2 and the increase in glycolysis induced by BRCA1 impairment. Evidence from this study indicates that the tumor suppressor functions of BRCA1 extend beyond DNA repair to include metabolic endpoints and identifies aspirin as an ovarian cancer chemopreventive agent capable of reversing the metabolic derangements caused by loss of BRCA1. Cancer Prev Res; 10(4); 255–66. ©2017 AACR.

m 6 A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer

N6-methyladenosine (m6A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m6A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m6A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in m6A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced m6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signalling.Liu et al. show that reduced m6A mRNA methylation in endometrial cancer is oncogenic. Mechanistically, the AKT pathway is activated in these tumours due to altered expression of AKT regulators carrying m6A on their transcripts.

An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells

Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Current methods limit the biological contexts that can be addressed due to sample homogenization, signal-averaging, and bias toward abundant proteins. Here we report a platform that integrates family-wide chemical probes with proximity-dependent oligonucleotide amplification and imaging to quantify enzyme activity in native contexts with high spatial resolution. Application of this method, activity-dependent proximity ligation (ADPL), to serine hydrolase and cysteine protease enzymes enables quantification of differential enzyme activity resulting from endogenous changes in localization and expression. In a competitive format, small-molecule target engagement with endogenous proteins in live cells can be quantified. Finally, retention of sample architecture enables interrogation of complex environments such as cellular co-culture and patient samples. ADPL should be amenable to diverse probe and protein families to detect active enzymes at scale and resolution out of reach with current methods.The interrogation of enzyme activity involves the ensemble averaging of many cells, loss of spatial relationships and is often biased to abundant proteins. Here the authors develop activity-dependent proximity ligation to quantify enzyme activity at the cellular and sub-cellular level in relevant biological contexts.

Abstract TMEM-118: ROLES OF T-LAK CELL-ORIGINATED KINASE (TOPK) AND THE MICROENVIRONMENT IN DRIVING OVARIAN CANCER PROGRESSION

Ovarian cancer (OvCa) therapy is confounded by late detection and a lack of effective therapeutics. After debulking surgery, most patients initially respond to platinum-based chemotherapeutics, however their tumors inevitably develop resistance and recur. Developing new treatments for OvCa will depend on a thorough understanding of OvCa biology. We recently identified T-LAK cell-originated kinase (TOPK), a serine-threonine kinase, as highly expressed in OvCa. A member of the cancer/testis antigen (CTA) family, TOPK is induced by mutant TP53 and is expressed in early, in situ serous tubal intraepithelial carcinoma lesions. Targeting TOPK with a small-molecule inhibitor in a mouse model of OvCa led to significant reductions in tumor burden. To better understand the interplay between TOPK and the tumor and stroma in ovarian cancer progression, we performed compartment-resolved, quantitative shotgun proteomics on microdissected tumor and stromal samples across a hypothetical progression series of ovarian cancer from in situ STIC to omental metastases. With a highly-sensitive platform, we were able to accurately quantify proteins across the progression series and confirmed expression of known tumor and stromal markers in the data set. Interestingly, we observed a conserved stromal response associated with metastasis. This signature included both genes known to play a role in cancer-associated fibroblasts, as well as proteins with no previously described roles in the tumor stroma. Many components of the signature are also representative of the previously described “mesenchymal” subgroup of ovarian cancer. In vitro and in vivo functional assays have confirmed essential roles for several of these proteins in regulating the cancer-associated fibroblast phenotype. This work establishes TOPK as a potential therapeutic target for ovarian cancer and emphasizes the importance of compartment resolved studies in understanding the individual roles of the tumor and stroma in ovarian cancer progression. Citation Format: Mark A. Eckert, Fabian Coscia, Shawn Pan, Chun-Yi Chiang, Stephanie McGregor, Ricardo Lastra, S. Diane Yamada, Matthias Mann, and Ernst Lengyel. ROLES OF T-LAK CELL-ORIGINATED KINASE (TOPK) AND THE MICROENVIRONMENT IN DRIVING OVARIAN CANCER PROGRESSION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-118.

Abstract 5899: Compartment-resolved proteomics reveal NNMT as a master metabolic regulator of cancer associated fibroblasts

The poor outcome of ovarian cancer patients is due to late detection and the ability of ovarian cancer cells to metastasize quickly throughout the abdominal cavity. Recent research, including histopathological studies and experiments with mouse models, suggest that ovarian cancer may actually arise in the fallopian tube as serous tubal intraepithelial carcinomas (STIC). Lesions of the ovary would, therefore, represent metastases from the fallopian tube. To better understand the molecular events that occur in both the tumor and stromal compartment during ovarian cancer progression, we performed shotgun proteomics on laser-microdissected tumor and stromal compartments from anatomic sites representing the hypothetical progression series for ovarian cancer (STIC → fallopian tube → ovary → omentum). With an optimized sample preparation technique, we successfully quantified 4-5000 proteins per anatomic site with high reproducibility and with as few as several thousand microdissected cells derived from formalin-fixed and paraffin-embedded (FFPE) biobank specimens. Proteomics of the tumor compartments revealed high inter-patient heterogeneity, with no conserved protein signatures associated with invasion or metastasis. In strong contrast, a highly conserved molecular signature of stromal proteins associated with metastasis to the omentum was identified. In particular, nicotinamide N-methyl transferase (NNMT) was highly upregulated in the stroma of omental and peritoneal metastases. Functionally, NNMT was necessary and sufficient for multiple aspects of the cancer associated fibroblast (CAF) phenotype, including expression of CAF markers and secretion of cytokines and oncogenic extracellular matrix. NNMT expression was necessary for the ability of CAFs to support ovarian cancer cell migration, proliferation, adhesion, and in vivo growth. Mechanistically, high expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics for compartment-resolved identification of candidate drivers of disease phenotypes and identifies NNMT as a central, metabolic regulator of CAF differentiation. Citation Format: Mark A. Eckert, Fabian Coscia, Shawn Pan, Samantha M. Tienda, Agnieszka A. Chryplewicz, Chun-Yi Chiang, Anthony Montag, S. Diane Yamada, Matthias Mann, Ernst R. Lengyel. Compartment-resolved proteomics reveal NNMT as a master metabolic regulator of cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5899. doi:10.1158/1538-7445.AM2017-5899