KLF3 and PAX6 are candidate driver genes in late-stage, MSI-hypermutated endometrioid endometrial carcinomas.

Endometrioid endometrial carcinomas (EECs) are the most common histological subtype of uterine cancer. Late-stage disease is an adverse prognosticator for EEC. The purpose of this study was to analyze EEC exome mutation data to identify late-stage-specific statistically significantly mutated genes (SMGs), which represent candidate driver genes potentially associated with disease progression. We exome sequenced 15 late-stage (stage III or IV) non-ultramutated EECs and paired non-tumor DNAs; somatic variants were called using Strelka, Shimmer, SomaticSniper and MuTect. Additionally, somatic mutation calls were extracted from The Cancer Genome Atlas (TCGA) data for 66 late-stage and 270 early-stage (stage I or II) non-ultramutated EECs. MutSigCV (v1.4) was used to annotate SMGs in the two late-stage cohorts and to derive p-values for all mutated genes in the early-stage cohort. To test whether late-stage SMGs are statistically significantly mutated in early-stage tumors, q-values for late-stage SMGs were re-calculated from the MutSigCV (v1.4) early-stage p-values, adjusting for the number of late-stage SMGs tested. We identified 14 SMGs in the combined late-stage EEC cohorts. When the 14 late-stage SMGs were examined in the TCGA early-stage data, only Krüppel-like factor 3 (KLF3) and Paired box 6 (PAX6) failed to reach significance as early-stage SMGs, despite the inclusion of enough early-stage cases to ensure adequate statistical power. Within TCGA, nonsynonymous mutations in KLF3 and PAX6 were, respectively, exclusive or nearly exclusive to the microsatellite instability (MSI)-hypermutated molecular subgroup and were dominated by insertions-deletions at homopolymer tracts. In conclusion, our findings are hypothesis-generating and suggest that KLF3 and PAX6, which encode transcription factors, are MSI target genes and late-stage-specific SMGs in EEC.

High-risk endometrial cancer proteomic profiling reveals that FBXW7 mutation alters L1CAM and TGM2 protein levels.

BACKGROUND: FBXW7 is frequently somatically mutated in grade 3 endometrioid endometrial cancers (G3EECs) and serous endometrial cancers (SECs), which are high-risk cancers associated with poor outcomes and in need of novel treatment options. The aim of this study was to determine the proteomic effects of 3 FBXW7 mutations in high-risk endometrial cancers (ECs). METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR) editing was used to generate 3 HEC-50B G3EEC derivative cell lines, each of which harbored 1 FBXW7 mutation, and to revert an endogenous FBXW7 mutation in HEC-1-B grade 2 endometrioid endometrial cancer (G2EEC) cells to the wild-type genotype. Proteomic profiling based on liquid chromatography-tandem mass spectrometry was used to determine protein differences between the HEC-50B derivative lines and parental cells. Western blot analysis was performed to assess differential protein levels of CRISPR-edited derivative lines originating from HEC-50B, ARK1 (SEC), ARK4 (SEC), HEC-1-B, and JHUEM-1 (G2EEC) cell lines in comparison with parental cells. RESULTS: Results of this study demonstrated the effects of FBXW7 mutations on the proteome and phosphoproteome of HEC-50B G3EEC cells and highlighted proteins that also exhibited altered levels in FBXW7-mutated ARK1 and ARK4 SEC cells, including 2 potentially druggable proteins: L1 cell adhesion molecule (L1CAM) and transglutaminase 2 (TGM2). Furthermore, they demonstrated that reversion of an endogenous FBXW7 mutation to the wild-type genotype in JHUEM-1 and HEC-1-B G2EEC cells resulted in decreased L1CAM and TGM2 protein levels. CONCLUSIONS: L1CAM and TGM2 protein levels are affected by FBXW7 mutations in ECs.

Proteomic profiling of FBXW7-mutant serous endometrial cancer cells reveals upregulation of PADI2, a potential therapeutic target.

BACKGROUND: Despite advancements over the past decade revealing molecular aberrations characteristic of endometrial cancer (EC) subtypes, serous ECs remain difficult to treat and associated with poor outcomes. This is due, in part, to the rarity of these tumors within clinical trials and the inability to directly target the most frequent genomic abnormalities. One of the most commonly somatically mutated genes in serous ECs is the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7). METHODS: To identify changes in protein expression associated with FBXW7 mutation, we clustered regularly interspaced short palindromic repeats (CRISPR)-edited ARK4 FBXW7 nonmutant serous EC cells to insert recurrent FBXW7 mutations. We then compared the liquid chromatography tandem mass spectrometry-based proteomic profiles of CRISPR-edited ARK1 and ARK4 serous EC cells to matched parental cells. RESULTS: Among distinct total and phosphorylated proteins that were significantly differentially expressed in FBXW7-mutant cell lines compared to matched parental lines, we identified increased PADI2 (peptidyl arginine deiminase 2) expression in all ARK1 and ARK4 CRISPR-edited FBXW7-mutant cell lines. We further confirmed the correlation between FBXW7 mutation and increased PADI2 expression in a third biological background, JHUEM-1 endometrioid EC cells. Finally, we established that PADI2 protein is expressed in primary serous endometrial tumors. CONCLUSION: Our findings provide novel insight into proteomic changes associated with FBXW7 mutation in serous ECs and identify PADI2 as a novel potential therapeutic target for these tumors.

Clinical actionability of molecular targets in endometrial cancer.

Endometrial cancer accounts for ~76,000 deaths among women each year worldwide. Disease mortality and the increasing number of new diagnoses make endometrial cancer an important consideration in women's health, particularly in industrialized countries, where the incidence of this tumour type is highest. Most endometrial cancers are carcinomas, with the remainder being sarcomas. Endometrial carcinomas can be classified into several histological subtypes, including endometrioid, serous and clear cell carcinomas. Histological subtyping is currently used routinely to guide prognosis and treatment decisions for endometrial cancer patients, while ongoing studies are evaluating the potential clinical utility of molecular subtyping. In this Review, we summarize the overarching molecular features of endometrial cancers and highlight recent studies assessing the potential clinical utility of specific molecular features for early detection, disease risk stratification and directing targeted therapies.

In vitro effects of FBXW7 mutation in serous endometrial cancer: Increased levels of potentially druggable proteins and sensitivity to SI-2 and dinaciclib.

Serous endometrial cancers (ECs) are clinically aggressive tumors that frequently harbor somatic mutations in FBXW7 (F-box and WD repeat domain-containing 7). The FBXW7 tumor suppressor is part of a SCF (complex of SKP1, Cullin 1, F-box protein) ubiquitin ligase complex which controls the degradation of numerous substrates that, if not properly regulated, can contribute to the initiation or progression of tumorigenesis. Despite reports that up to 30% of serous ECs include somatic mutations in FBXW7, the molecular effects of mutated FBXW7 in ECs have not been determined. Here, we used transient transfection and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing in serous EC cell lines to interrogate the molecular effects of six recurrent FBXW7 mutations. We show that FBXW7 mutations lead to increased Cyclin E1, steroid receptor coactivator 3 (SRC-3), c-MYC, Rictor, glycogen synthase kinase 3 (GSK3), P70S6 kinase, and protein kinase B (AKT) phosphorylated protein levels in serous EC cells. Furthermore, we demonstrate that CRISPR-edited FBXW7-mutant ARK1 serous EC cells exhibit increased sensitivity to SI-2 (a SRC inhibitor) and dinaciclib (a cyclin dependent kinase (CDK) inhibitor) compared to parental ARK1 cells. Collectively, our findings reveal biochemical effects of FBXW7 mutations in the context of EC and provide in vitro evidence of sensitivity to targeted inhibitors.

The FOXA2 transcription factor is frequently somatically mutated in uterine carcinosarcomas and carcinomas.

BACKGROUND: Uterine carcinosarcomas (UCSs) are a rare but clinically aggressive form of cancer. They are biphasic tumors consisting of both epithelial and sarcomatous components. The majority of uterine carcinosarcomas are clonal, with the carcinomatous cells undergoing metaplasia to give rise to the sarcomatous component. The objective of the current study was to identify novel somatically mutated genes in UCSs. METHODS: We whole exome sequenced paired tumor and nontumor DNAs from 14 UCSs and orthogonally validated 464 somatic variants using Sanger sequencing. Fifteen genes that were somatically mutated in at least 2 tumor exomes were Sanger sequenced in another 39 primary UCSs. RESULTS: Overall, among 53 UCSs in the current study, the most frequently mutated of these 15 genes were tumor protein p53 (TP53) (75.5%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (34.0%), protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A) (18.9%), F-box and WD repeat domain containing 7 (FBXW7) (18.9%), chromodomain helicase DNA binding protein 4 (CHD4) (17.0%), and forkhead box A2 (FOXA2) (15.1%). FOXA2 has not previously been implicated in UCSs and was predominated by frameshift and nonsense mutations. One UCS with a FOXA2 frameshift mutation expressed truncated FOXA2 protein by immunoblotting. Sequencing of FOXA2 in 160 primary endometrial carcinomas revealed somatic mutations in 5.7% of serous, 22.7% of clear cell, 9% of endometrioid, and 11.1% of mixed endometrial carcinomas, the majority of which were frameshift mutations. CONCLUSIONS: Collectively, the findings of the current study provide compelling genetic evidence that FOXA2 is a pathogenic driver gene in the etiology of primary uterine cancers, including UCSs. Cancer 2018;124:65-73. © 2017 American Cancer Society.

Somatic mutation profiles of clear cell endometrial tumors revealed by whole exome and targeted gene sequencing.

BACKGROUND: The molecular pathogenesis of clear cell endometrial cancer (CCEC), a tumor type with a relatively unfavorable prognosis, is not well defined. We searched exome-wide for novel somatically mutated genes in CCEC and assessed the mutational spectrum of known and candidate driver genes in a large cohort of cases. METHODS: We conducted whole exome sequencing of paired tumor-normal DNAs from 16 cases of CCEC (12 CCECs and the CCEC components of 4 mixed histology tumors). Twenty-two genes-of-interest were Sanger-sequenced from another 47 cases of CCEC. Microsatellite instability (MSI) and microsatellite stability (MSS) were determined by genotyping 5 mononucleotide repeats. RESULTS: Two tumor exomes had relatively high mutational loads and MSI. The other 14 tumor exomes were MSS and had 236 validated nonsynonymous or splice junction somatic mutations among 222 protein-encoding genes. Among the 63 cases of CCEC in this study, we identified frequent somatic mutations in TP53 (39.7%), PIK3CA (23.8%), PIK3R1 (15.9%), ARID1A (15.9%), PPP2R1A (15.9%), SPOP (14.3%), and TAF1 (9.5%), as well as MSI (11.3%). Five of 8 mutations in TAF1, a gene with no known role in CCEC, localized to the putative histone acetyltransferase domain and included 2 recurrently mutated residues. Based on patterns of MSI and mutations in 7 genes, CCEC subsets molecularly resembled serous endometrial cancer (SEC) or endometrioid endometrial cancer (EEC). CONCLUSION: Our findings demonstrate molecular similarities between CCEC and SEC and EEC and implicate TAF1 as a novel candidate CCEC driver gene. Cancer 2017;123:3261-8. © 2017 American Cancer Society.

Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancer uncovers rare somatic mutations in TNK2 and DDR1.

BACKGROUND: Endometrial cancer (EC) is the 8th leading cause of cancer death amongst American women. Most ECs are endometrioid, serous, or clear cell carcinomas, or an admixture of histologies. Serous and clear ECs are clinically aggressive tumors for which alternative therapeutic approaches are needed. The purpose of this study was to search for somatic mutations in the tyrosine kinome of serous and clear cell ECs, because mutated kinases can point to potential therapeutic targets. METHODS: In a mutation discovery screen, we PCR amplified and Sanger sequenced the exons encoding the catalytic domains of 86 tyrosine kinases from 24 serous, 11 clear cell, and 5 mixed histology ECs. For somatically mutated genes, we next sequenced the remaining coding exons from the 40 discovery screen tumors and sequenced all coding exons from another 72 ECs (10 clear cell, 21 serous, 41 endometrioid). We assessed the copy number of mutated kinases in this cohort of 112 tumors using quantitative real time PCR, and we used immunoblotting to measure expression of these kinases in endometrial cancer cell lines. RESULTS: Overall, we identified somatic mutations in TNK2 (tyrosine kinase non-receptor, 2) and DDR1 (discoidin domain receptor tyrosine kinase 1) in 5.3% (6 of 112) and 2.7% (3 of 112) of ECs. Copy number gains of TNK2 and DDR1 were identified in another 4.5% and 0.9% of 112 cases respectively. Immunoblotting confirmed TNK2 and DDR1 expression in endometrial cancer cell lines. Three of five missense mutations in TNK2 and one of two missense mutations in DDR1 are predicted to impact protein function by two or more in silico algorithms. The TNK2(P761Rfs*72) frameshift mutation was recurrent in EC, and the DDR1(R570Q) missense mutation was recurrent across tumor types. CONCLUSIONS: This is the first study to systematically search for mutations in the tyrosine kinome in clear cell endometrial tumors. Our findings indicate that high-frequency somatic mutations in the catalytic domains of the tyrosine kinome are rare in clear cell ECs. We uncovered ten new mutations in TNK2 and DDR1 within serous and endometrioid ECs, thus providing novel insights into the mutation spectrum of each gene in EC.

MEK1/2 inhibition enhances the radiosensitivity of cancer cells by downregulating survival and growth signals mediated by EGFR ligands.

The inhibition of the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway through the suppression of mutated Ras or MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) has been shown to sensitize tumor cells to ionizing radiation (IR). The molecular mechanisms of this sensitization however, are not yet fully understood. In this study, we investigated the role of transforming growth factor-α (TGF-α) in the radiosensitizing effects of selumetinib, a selective inhibitor of MEK1/2. The expression of epidermal growth factor receptor (EGFR) ligands was assessed by ELISA in both Ras wild-type and Ras mutant cells that were exposed to radiation with or without selumetinib. The effects of selumetinib on the TGF-α/EGFR signaling cascade in response to radiation were examined by western blot analysis, clonogenic assay and by determing the yield of mitotic catastrophe. The treatment of cells with selumetinib reduced the basal and IR-induced secretion of TGF-α in both Ras wild-type and Ras mutant cell lines in vitro and in vivo. The reduction of TGF-α secretion was accompanied with a reduction in phosphorylated tumor necrosis factor-α converting enzyme (TACE) in the cells treated with selumetinib with or without IR. The treatment of cells with selumetinib with or without IR inhibited the phosphorylation of EGFR and checkpoint kinase 2 (Chk2), and reduced the expression of survivin. Supplementation with exogenous TGF-α partially rescued the selumetinib-treated cells from IR-induced cell death, restored EGFR and Chk2 phosphorylation and increased survivin expression. These data suggest that the inhibition of MEK1/2 with selumetinib may provide a mechanism to sensitize tumor cells to IR in a fashion that prevents the activation of the TGF-α autocrine loop following IR.

Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes.

Endometrial cancer is the sixth most commonly diagnosed cancer in women worldwide, causing ~74,000 deaths annually. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology. We used whole-exome sequencing to comprehensively search for somatic mutations within ~22,000 protein-encoding genes in 13 primary serous endometrial tumors. We subsequently resequenced 18 genes, which were mutated in more than 1 tumor and/or were components of an enriched functional grouping, from 40 additional serous tumors. We identified high frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%). Overall, 36.5% of serous tumors had a mutated chromatin-remodeling gene, and 35% had a mutated ubiquitin ligase complex gene, implicating frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.

Determination of cytokine protein levels in oral secretions in patients undergoing radiotherapy for head and neck malignancies.

BACKGROUND: Cytokines may be elevated in tumor and normal tissues following irradiation. Cytokine expression in these tissues may predict for toxicity or tumor control. The purpose of this pilot study was to determine the feasibility of measuring local salivary cytokine levels using buccal sponges in patients receiving chemo-radiation for head and neck malignancies. PATIENTS AND METHODS: 11 patients with epithelial malignancies of the head and neck were recruiting to this study. All patients received radiotherapy to the head and neck region with doses ranging between 60 - 67.5 Gy. Chemotherapy was delivered concurrently with radiation in all patients. Salivary samples were obtained from high dose and low dose regions prior to treatment and at three intervals during treatment for assessment of cytokine levels (IL-4, IL-6, IL-8, IL-10, EGF, MCP-1, TNF-α, and VEGF). RESULTS: Cytokine levels were detectable in the salivary samples. Salivary cytokine levels of IL-4, IL-6, IL-8, EGF, MCP-1, TNF- α , and VEGF were higher in the high dose region compared to the low dose region at all time points (p < 0.05). A trend toward an increase in cytokine levels as radiation dose increased was observed for IL-6, IL-8, MCP-1, and TNF-α. CONCLUSION: Assessment of salivary cytokine levels may provide a novel method to follow local cytokine levels during radiotherapy and may provide a mechanism to study cytokine levels in a regional manner.

Enhancement of 5-fluorouracil-induced in vitro and in vivo radiosensitization with MEK inhibition.

PURPOSE: Gastrointestinal cancers frequently exhibit mutational activation of the Ras/MAPK pathway, which is implicated in resistance to ionizing radiation (IR) and chemotherapy. Concurrent radiotherapy and 5-fluorouracil (5-FU) based chemotherapy is commonly used for treatment of gastrointestinal malignancies. We previously reported radiosensitization with selumetinib, an inhibitor of MEK1/2. The purpose of the current study was to evaluate if selumetinib could enhance radiosensitivity induced by 5-FU. EXPERIMENTAL DESIGN: Clonogenic survival assays were carried out with the HT29 (colorectal), HCT116 (colorectal), and MiaPaca-2 (pancreatic) cell lines using pre-IR treatment with selumetinib, 5-FU and 5-FU+selumetinib. Cell proliferation was determined using a tetrazolium conversion assay. Mitotic catastrophe and DNA repair were analyzed using immunocytochemistry. Flow cytometry was used to analyze cell cycle and apoptosis. Growth delay was used to determine effects of 5-FU+selumetinib on in vivo tumor radiosensitivity. RESULTS: Pre-IR treatment with 5-FU+selumetinib significantly decreased clonogenic survival compared with either agent alone. Dose modifying factors at a surviving fraction of 0.1 for 5-FU+selumetinib was 1.78, 1.52, and 1.3 for HT29, HCT116, and MiaPaca-2, respectively. Cell proliferation was decreased by treatment with selumetinib+5-FU as compared with single agent treatment regardless of treatment sequencing. Enhancement of 5-FU cytotoxicity and 5-FU mediated radiosensitization with selumetinib treatment was accompanied by an increase in mitotic catastrophe and apoptosis, and reductions in Stat3 phosphorylation and survivin expression. In vivo, an additive growth delay was observed with 5-FU+selumetinib+3Gy versus 5-FU+3Gy and selumetinib alone. CONCLUSION: These data suggest that selumetinib can be used with 5-FU to augment radiation response.

Evaluation of the fullerene compound DF-1 as a radiation protector.

BACKGROUND: Fullerene compounds are known to possess antioxidant properties, a common property of chemical radioprotectors. DF-1 is a dendrofullerene nanoparticle with antioxidant properties previously found to be radioprotective in a zebrafish model. The purpose of this study was to evaluate the radioprotective effects of DF-1 in a murine model of lethal total body irradiation and to assess for selective radioprotection of normal cells versus tumor cells. METHODS: In vitro radioresponse was evaluated with clonogenic assays with human tumor cells and fibroblast lines in the presence of varying concentrations of DF-1 or vehicle. DNA double strand break induction and repair was evaluated with immunocytochemistry for gammaH2AX. Lethal total body irradiation was delivered with 137Cs after intraperitoneal delivery of DF-1 or vehicle control. Bone marrow hypoxia was evaluated with piminidazole uptake assessed by flow cytometry. RESULTS: DF-1 provided modest radioprotection of human cancer cell lines and fibroblast cell lines when delivered prior to irradiation (dose modifying factor or 1.1). There was no evidence of selective protection of fibroblasts versus tumor cells. Cells treated with DF-1 at radioprotective doses were found to have fewer gammaH2AX foci at 1 and 6 hours after irradiation compared to vehicle treated controls. The LD50/30 for C57Bl6/Ncr mice treated with a single 300 mg/kg dose of DF-1 pre-irradiation was 10.09 Gy (95% CI 9.58-10.26) versus 8.29 Gy (95% CI, 8.21-8.32) for control mice. No protective effects were seen with a single 200 mg/kg dose. No increase in pimonidazole uptake was appreciated in bone marrow of mice treated with DF-1 compared to vehicle controls. CONCLUSIONS: DF-1 has modest activity as a radiation protector in vivo. There was no evidence of selective protection from irradiation of normal versus tumor cells with DF-1.

Gene expression profiling reveals differentially expressed genes in ovarian cancer of the hen: support for oviductal origin?

Ovarian cancer has a high mortality rate due, in part, to the lack of early detection and incomplete understanding of the origin of the disease. The hen is the only spontaneous model of ovarian cancer and can therefore aid in the identification and testing of early detection strategies and therapeutics. Our aim was to combine the use of the hen animal model and microarray technology to identify differentially expressed genes in ovarian tissue from normal hens compared with hens with ovarian cancer. We found that the transcripts up-regulated in chicken ovarian tumors were enriched for oviduct-related genes. Quantitative real-time PCR and immunohistochemistry confirmed expression of oviduct-related genes in normal oviduct and in ovaries from hens with early- and late-stage ovarian tumors, but not in normal ovarian surface epithelium. In addition, one of the oviduct-related genes identified in our analysis, paired box 2 has been implicated in human ovarian cancer and may serve as a marker of the disease. Furthermore, estrogen receptor 1 mRNA is over-expressed in early-stage tumors, suggesting that expression of the oviduct-related genes may be regulated by estrogen. We have also identified oviduct-related genes that encode secreted proteins that could represent putative serum biomarkers. The expression of oviduct-related genes in early-stage tumors is similar to what is seen in human ovarian cancer, with tumors resembling normal Müllerian epithelium. These data suggest that chicken ovarian tumors may arise from alternative sites, including the oviduct.