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Summary Pathological examination is the gold standard for cancer diagnosis, and breast tumor cells are often found in clusters. We report a case study on one triple-negative breast cancer (TNBC) patient, analyzing tumor development, metastasis, and prognosis with simultaneous DNA and RNA sequencing of pathologist-defined cell clusters from multiregional frozen sections. The cell clusters are isolated by laser capture microdissection (LCM) from primary tumor tissue, lymphatic vessels, and axillary lymph nodes. Data are reported for a total of 97 cell clusters. A combination of tumor cell-cluster clonality and phylogeny reveals 3 evolutionarily distinct pathways for this patient, each associated with a unique mRNA signature, and each correlated with disparate survival outcomes. Hub gene analysis indicates that extensive downregulation of ribosomal protein mRNA is a potential marker of poor prognosis in breast cancer., Graphical abstract, Highlights Pathologically diverse cell clusters share genomic and transcriptomic profiles Transcriptome-defined clones are more complex than genome-defined clones Three distinct pathways were inferred, each with disparate survival outcomes Lower expression of ribosomal proteins may be an indicator of poor prognosis, Zhu et al. reveal the complexity of tumor development, metastasis, and prognosis by simultaneous DNA and RNA sequencing of pathologist-defined cell clusters, excised through laser capture microdissection from multiregional frozen sections of a triple-negative breast cancer patient.

The objective of this study was to investigate if DHA dietary supplementation enhances the anticancer actions of docetaxel (TXT) in two different drug resistant triple negative breast cancer (TNBC) patient-derived xenografts (PDX)s. In two experiments, female NSG mice bearing TNBC PDXs were randomized to one of two nutritionally adequate diets (20% w/w): control (0% DHA), or DHA (3.9% w/w of total fat) and injected with 0 or 5 mg/kg TXT, twice weekly for 6 weeks (n = 8 per group). Treatment response was determined by significant differences in tumor weight, and apoptotic, proliferation and cell cycle markers at endpoint. Mice bearing MAXF574 xenografts fed DHA diet and treated with TXT had a 57% reduction in tumor weight compared to mice fed control diet (P

Simple Summary There has been extensive research into the beneficial anticancer effects of n-3 long-chain polyunsaturated fatty acids (LCPUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in preclinical models of cancer. However, clinical evidence is limited. The aim of this scoping review was to summarize the current clinical evidence of n-3 LCPUFA supplementation in cancer treatment and highlight areas where more clinical evidence is needed. We summarized the results of 57 clinical trials with an EPA/DHA intervention and determined that supplementation could improve a variety of outcomes important to the patient and the disease process, including immune system modulation, improved weight maintenance and increased disease-free or progression-free survival. There is, however, a need for larger, well-controlled, statistically powered randomized controlled trials to move n-3 supplementation to clinical practice. Abstract This scoping review examines the evidence for n-3 long-chain polyunsaturated fatty acid [LCPUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] supplementation in clinical cancer therapy. A comprehensive literature search was performed to identify relevant clinical intervention studies conducted through August 2020. Fifty-seven unique cancer trials, assessing EPA and/or DHA supplementation pre- or post-treatment, concomitant with neoadjuvant chemotherapy, radiation or surgery, or in palliative therapy were included. Breast, head and neck, gastrointestinal, gastric, colorectal/rectal, esophageal, leukemia/lymphoma, lung, multiple myeloma and pancreatic cancers were investigated. Across the spectrum of cancers, the evidence suggests that supplementation increased or maintained body weight, increased progression-free and overall survival, improved overall quality of life, resulted in beneficial change in immune parameters and decreased serious adverse events. Taken together, the data support that EPA and/or DHA could be used to improve outcomes important to the patient and disease process. However, before incorporation into treatment can occur, there is a need for randomized clinical trials to determine the dose and type of n-3 LCPUFA intervention required, and expansion of outcomes assessed and improved reporting of outcomes.

Dedifferentiated/undifferentiated endometrial carcinoma (DDEC/UEC) is an endometrial cancer characterized by the presence of histologically undifferentiated carcinoma. Genomic inactivation of core switch/sucrose nonfermentable (SWI/SNF) complex proteins was recently identified in approximately two‐thirds of DDEC/UEC. The aim of this study was to delineate the clinical behavior of SWI/SNF‐deficient DDEC/UEC in comparison to SWI/SNF‐intact DDEC/UEC. The study cohort consisted of 56 SWI/SNF‐deficient DDEC/UEC (2 POLE‐mutated), which showed either SMARCA4 (BRG1) loss, ARID1A/1B co‐loss, or SMARCB1 (INI1) loss in the undifferentiated tumor, and 26 SWI/SNF‐intact DDEC/UEC (4 POLE‐mutated). The average age at diagnosis was 61 years for patients with SWI/SNF‐deficient tumors and 64 years for SWI/SNF‐intact tumors. Mismatch repair (MMR) protein deficiency was seen in 66% of SWI/SNF‐deficient and 50% of SWI/SNF‐intact tumors. At initial presentation, 55% of patients with SWI/SNF‐deficient tumors had extrauterine disease spread in contrast to 38% of patients with SWI/SNF‐intact tumors. The 2‐year disease specific survival (DSS) for stages I and II disease was 65% for SWI/SNF deficient tumors relative to 100% for SWI/SNF‐intact tumors (p = 0.042). For patients with stages III and IV disease, the median survival was 4 months for SWI/SNF‐deficient tumors compared to 36 months for SWI/SNF‐intact tumors (p = 0.0003). All six patients with POLE‐mutated tumors, including one with stage IV SWI/SNF‐deficient tumor were alive with no evidence of disease. Among the patients with advanced stage SWI/SNF‐deficient tumors, 68% (21 of 31) received adjuvant or neoadjuvant chemotherapy (platinum/taxane‐based) and all except the patient with a POLE‐mutated tumor (20 of 21) experienced disease progression either during chemotherapy or within 4 months after its completion. These findings show that core SWI/SNF‐deficiency defines a highly aggressive group of undifferentiated cancer characterized by rapid disease progression that is refractory to conventional platinum/taxane‐based chemotherapy. This underscores the importance of accurate clinical recognition of this aggressive tumor and the need to consider alternative systemic therapy for these tumors.

Background: N-terminal myristoylation is the transfer of the saturated fourteen carbon fatty acid myristate to an N-terminal glycine residue. This co- or post-translational protein modification promotes protein-protein and protein-lipid interactions and is essential for proper membrane localization and/or activity of up to 600 human intracellular proteins. N-myristoyltransferases (NMTs) are the enzymes responsible and two isoforms are found in humans. NMT1 (ubiquitous and essential for cell survival) and NMT2 (more variably expressed) differ in activity level and substrate specificities. NMT expression levels vary in some cancers, and with myristoylation being essential for activity of certain oncogenes including Src Family Kinases (SFKs). NMTs have therefore been proposed as an anti-cancer target. Dysregulation and oncogenic activity of SFKs occurs frequently in acute myeloid leukemia (AML), suggesting NMT inhibition could provide therapeutic benefit. PCLX-001 is a low nanomolar small molecule pan-NMT inhibitor with high oral bioavailability in clinical trials as once daily oral therapy for lymphoma and solid tumors. Methods and Results: Data from the TCGA Transcriptome database showed high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, as well as reduced levels of SFK proteins and SFK phosphorylation. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations which spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. AML cell lines showed significant increase in BIP protein and ER stress in response to PCLX-001, along with caspase 3 cleavage. In an AML cell line derived xenograft (CDX) and two AML patient derived xenograft (PDX) series (n=1 DX for MV-4-11 and n=2 PDX), PCLX-001 monotherapy had dose-dependent anticancer activity and resulted in complete remissions in subcutaneous AML cell deposits. In tail-vein injection PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. Conclusions: These findings validate NMT inhibition as a novel therapeutic strategy for AML. PCLX-001 preferentially targeted AML cells that rely on oncogenic activity of myristoylated proteins, inducing apoptosis and reducing leukemic burden. PCLX-001 warrants evaluation in clinical trials for adult AML. Disclosures Gamma: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company. Yap: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company, Patents & Royalties. Beauchamp: Pacylex Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company, Patents & Royalties. Mackey: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company. Pemmaraju: Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Aptitude Health: Consultancy; Sager Strong Foundation: Other; Celgene Corporation: Consultancy; LFB Biotechnologies: Consultancy; Plexxicon: Other, Research Funding; MustangBio: Consultancy, Other; Roche Diagnostics: Consultancy; Daiichi Sankyo, Inc.: Other, Research Funding; DAVA Oncology: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Springer Science + Business Media: Other; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; CareDx, Inc.: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Borthakur: Astex: Research Funding; Ryvu: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Brandwein: AbbVie: Honoraria; Jazz: Honoraria; Taiho: Honoraria; Astellas: Honoraria; Bristol Myers Squibb: Honoraria; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Berthiaume: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company.

Dedifferentiated carcinoma of the endometrium or the ovary is an aggressive epithelial malignancy that comprises an endometrioid carcinoma together with an undifferentiated carcinoma. We recently reported that inactivation of BRG1 or INI1, core subunits of the switch/sucrose non-fermenting (SWI/SNF) complex, was the likely molecular event underlying dedifferentiation in about half of dedifferentiated carcinomas. In this study, we performed a genomic screen that included other members of the SWI/SNF complex to better delineate the molecular basis in the remainder of these tumours. We identified concurrent inactivating mutations involving ARID1A and ARID1B in 12 of 24 BRG1/INI1-intact, 0 of 3 INI1-deficient and 0 of 16 BRG1-deficient dedifferentiated carcinomas. All ARID1A and ARID1B co-mutated tumours displayed loss of ARID1A expression in the undifferentiated component with 11 of 12 tumours also displaying absent staining in the endometrioid component. ARID1B expression was absent in the undifferentiated component in all 12 tumours, whereas the corresponding endometrioid component showed intact expression. Clinically, ARID1A/ARID1B co-inactivated tumours showed similar aggressive behaviour to BRG1 or INI1-inactivated tumours. Given that ARID1A and ARID1B are the only known DNA-binding subunits of the SWI/SNF-A complex, additional inactivation of ARID1B in an ARID1A-deficient background appears to represent an alternative mechanism of disruption of SWI/SNF-mediated transcriptional regulation, resulting in arrested cellular differentiation in endometrial and ovarian endometrioid cancer.

Myristoylation is the N-terminal modification of proteins with the fatty acid myristate. This process is mediated by two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, and is critical for membrane targeting and cell signaling. Because NMT expression is increased in some cancers, we used three robotic screens to evaluate the potential of the potent pan-NMT inhibitor PCLX-001 on 300 cancer cell lines spanning the spectrum of human cancers. We discovered a marked increase in the sensitivity of hematological cancer cell lines, including B-cell lymphomas, to myristoylation inhibition. PCLX-001 consistently reduced both lymphoma cell proliferation and viability at concentrations lower than those needed to inhibit the growth of or to kill benign immortalized B cells. In lymphoma cell lines, PCLX-001 treatment inhibited early B-cell receptor (BCR) signaling events by disrupting membrane targeting of several myristoylated Src family kinases and promoted their ubiquitin-mediated degradation. Unexpectedly, PCLX-001 also promoted the degradation of non-myristoylated transcriptional activators P-ERK, c-Myc, NFκB and CREB downstream in the BCR signaling cascade, leading to loss of survival signals and apoptosis. Furthermore, compared to clinically approved drugs dasatinib and ibrutinib, PCLX-001 was more potent in vitro at inhibiting B-cell signaling, had a wider breadth of efficacy, and had greater selectivity thus sparing normal B cells. PCLX-001 treatment reduced tumor size in a time and concentration dependent manner in three B-cell lymphoma xenograft models and resulted in complete disease regression in two of these models, including an R-CHOP refractory lymphoma patient-derived xenograft. To investigate the potential mechanisms responsible for the sensitivity of hematological cancers to PCLX-001, we examined the NMT expression levels in cancer cells using publically available databases. Contrary to the reported NMT overexpression in some cancers, we found that hematological cancer cell lines and tumors both display significant reduction in NMT2 expression. The decreased NMT2 expression is significantly correlated with lower EC50 and poorer patient prognosis. Using the CRISPR-based genetic alteration Cancer Dependency Map, we discovered that cancer cells are highly dependent on functional NMT1, and that NMT1 dependency increases with low NMT2 expression. PCLX-001 treatment may mimic the effect of genetic alteration of NMT1 in hematological cancer cells low in NMT2 by pharmacologically inhibiting the remaining NMT1 in these cells. This results in an effect reminiscent of synthetic lethality since the vast majority of normal cells express both NMTs and PCLX-001 selectively kills NMT2-deficient cancer cells while sparing normal cells. Our findings support the ongoing development and eventual clinical trials of PCLX-001 as a therapy for hematological cancers. Citation Format: Erwan Beauchamp, Megan C. Yap, Maneka A. Perinpanayagam, Jay M. Gamma, Krista M. Vincent, Raymond Lai, Wei-Feng Dong, Manikandan Lakshmanan, Anandhkumar Raju, Vinay Tergaonkar, Soo Yong Tan, Soon Thye Lim, Lynne Postovit, Kevin D. Read, David W. Gray, Paul G. Wyatt, John R. Mackey, Luc G. Berthiaume. Targeting N-myristoylation in B-cell lymphomas as a therapeutic strategy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5156.

NODALis a morphogen essential for early embryonic development in vertebrates. Since much of our understanding ofNODALcomes from model organisms, we aimed to directly assess post-transcriptional regulation of human NODAL with specific attention to a newly discovered human-specific NODAL splice variant. Selective depletion of the NODAL variant in human embryonic stem cells resulted in increased LIFR levels, while total NODAL knockdown resulted in a decrease of several markers of pluripotency. The NODAL variant did not transmit a canonical NODAL signal in zebrafish embryos, but may share some functional capability with canonical NODAL in cancer cells. At the protein level, disruption of disulfide bond formation dramatically enhanced proteolytic processing of NODAL. Disruption of NODAL N-glycosylation decreased its secretion but not extracellular stability, and a novel N-glycosylation in the NODAL variant contributed to enhanced secretion. Collectively, this work offers a direct and precise account of post-transcriptional regulation of human NODAL.

Globally, there were 14.1 million new cancer diagnoses and 8.2 million cancer deaths in 2012. For many cancers, conventional therapies are limited in their successes and an improved understanding of disease progression is needed in conjunction with exploration of alternative therapies. The long chain polyunsaturated fatty acid, docosahexaenoic acid (DHA), has been shown to enhance many cellular responses that reduce cancer cell viability and decrease proliferation both in vitro and in vivo. A small number of studies suggest that DHA improves chemotherapy outcomes in cancer patients. It is readily incorporated into cancer cell membranes and, as a result there has been considerable research regarding cell membrane initiated events. For example, DHA has been shown to mediate the induction of apoptosis/reduction of proliferation in vitro and in vivo. However, there is limited research into the effect of DHA on cell cycle regulation in cancer cells and the mechanism(s) by which DHA acts are not fully understood. The purpose of the current review is to provide a critical examination of the literature investigating the ability of DHA to stall progression during different cell cycle phases in cancer cells, as well as the consequences that these changes may have on tumour growth, independently and in conjunction with chemotherapy.

IntroductionNeoadjuvant chemotherapy for breast cancer treatment is prescribed to facilitate surgery and provide confirmation of drug-sensitive disease, and the achievement of pathological complete response (pCR) predicts improved long-term outcomes. Docosahexaenoic acid (DHA) has been shown to reduce tumour growth in preclinical models when combined with chemotherapy and is known to beneficially modulate systemic immune function. The purpose of this trial is to investigate the benefit of DHA supplementation in combination with neoadjuvant chemotherapy in patients with breast cancer.Methods and analysisThis is a double-blind, phase II, randomised controlled trial of 52 women prescribed neoadjuvant chemotherapy to test if DHA supplementation enhances chemotherapy efficacy. The DHA supplementation group will take 4.4 g/day DHA orally, and the placebo group will take an equal fat supplement of vegetable oil. The primary outcome will be change in Ki67 labelling index from prechemotherapy core needle biopsy to definitive surgical specimen. The secondary endpoints include assessment of (1) DHA plasma phospholipid content; (2) systemic immune cell types, plasma cytokines and inflammatory markers; (3) tumour markers for apoptosis and tumour infiltrating lymphocytes; (4) rate of pCR in breast and in axillary nodes; (5) frequency of grade 3 and 4 chemotherapy-associated toxicities; and (6) patient-perceived quality of life. The trial has 81% power to detect a significant between-group difference in Ki67 index with a two-sided t-test of less than 0.0497, and accounts for 10% dropout rate.Ethics and disseminationThis study has full approval from the Health Research Ethics Board of Alberta - Cancer Committee (Protocol #: HREBA.CC-18-0381). We expect to present the findings of this study to the scientific community in peer-reviewed journals and at conferences. The results of this study will provide evidence for supplementing with DHA during neoadjuvant chemotherapy treatment for breast cancer.Trial registration numberNCT03831178

Treatment of aggressive lymphoma is toxic, expensive, and a substantial proportion of patients relapse and die. There is an urgent need for more effective treatments. Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. Since they are critical to intracellular signaling, NMTs are potential anti-cancer targets. We tested a novel potent pan-NMT inhibitor, PCLX-001, in B cell lymphoma cell lines. In vitro assays included cell viability, immunoblotting, and metabolic labeling of lymphoma cell lines. Immunohistochemistry was performed on formalin fixed paraffin embedded lymphoma specimens from patients. In vivo experiments included cell line derived murine xenografts and a patient derived mouse xenograft treated with increasing concentrations of PCLX-001. PCLX-001 selectively killed lymphoma cells, while sparing normal cells in vitro and in 3 mouse xenograft models, eradicating tumors in two of these models including a patient-derived xenograft from a R-CHOP refractory lymphoma patient. While NMT2 is overexpressed in some cancers, loss of NMT2 expression is common in numerous cancers and occurs at the highest prevalence in lymphomas, where it is independently linked to a worse prognosis. This NMT2 suppression occurred through epigenetic mechanisms and may account for lymphoma sensitivity to NMT inhibition. The global myristoylation of lymphoma cell proteins, including that of the protein tyrosine kinase oncogene Src, is profoundly inhibited by PCLX-001. Loss of Src myristoylation is accompanied by loss of Src activity and may account for loss of prosurvival signals causing lymphoma cell death. Targeting NMT2 deficient B cell lymphoma with a pan-NMT inhibitor suppresses the residual NMT1 function provides a novel, selective, and effective therapeutic strategy. Citation Format: John R. Mackey, Erwan Beauchamp, Megan C. Yap, Aishwarya Iyer, Maneka A. Perinpanayagam, Krista M. Vincent, Abass M. Al-Momany, Ryan J. Heit, Jacky Y. Sim, Raymond Lai, Wei-feng Dong, Manikandan Lakshmanan, Anandhkumar Raju, Vinay Tergaonkar, Soo Yong Tan, Soon Thye Lim, Lynne M. Postovit, Kevin D. Read, David W. Gray, Paul G. Wyatt, Luc G. Berthiaume. Targeting N-myristoylation in B cell lymphomas as a therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3046.

Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. In acute myeloid leukemia (AML), upregulation of Lyn and Src, important myristoylated proteins, contribute to cell survival and proliferation. The specific roles of NMT1 and NMT2 are unknown in this context. The relationships among NMT1/NMT2 expression and acute AML patient outcomes were studied using RNA-sequencing and microarray cohorts with over 350 patients. We found that high NMT1 and low NMT2 expression were associated with reduced overall and event-free survival in adult AML, which was independent of other prognostic markers on multivariate analysis. High NMT1, but not NMT2, expression was associated with proliferative gene sets in AML cell lines, indicating potential for distinct isozyme substrates. Given these results, we examined NMT1 and NMT2 levels in AML cell lines and AML patient blast cells using Western blotting and flow cytometry. We determined that NMT2 expression varied greatly among patients, but was markedly reduced in most cases, while NMT1 expression was relatively preserved. A potent small molecule NMT inhibitor, PCLX-001, preferentially induced apoptosis and reduced viability in NMT2-deficient AML cell lines cultured in vitro compared with normal lymphocytes and peripheral blood mononuclear cells. PCLX-001 also killed freshly isolated human AML blasts ex vivo with an IC50 of ~170nM regardless of their mutational background. In a murine AML xenograft model, subcutaneously delivered PCLX-001 monotherapy demonstrated dose-dependent anticancer activity and produced complete remissions after five daily 50 mg/kg doses. NMT expression provides independent prognostic information to refine existing clinical stratification, and NMT inhibition is a promising novel therapeutic strategy for AML. Citation Format: John R. Mackey, Aishwarya Iyer, Megan C. Yap, Zoulika Zak, Krista Vincent, Erwan Beauchamp, Lynne M. Postovit, Joseph Brandwein, Luc G. Berthiaume. Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3043.

Epigenetic silencing of RASSF1A is frequently observed in numerous cancers and has been previously reported. The promoter region of RASSF1A is predicted to have 75 CpG sites, and very few studies demonstrate how the methylation of these sites affects expression. In addition, the expression relationship between RASSF1A and its downstream target, modulator of apoptosis 1 (MOAP-1), is poorly understood. In this study, we have explored the mRNA expression of RASSF1A, MOAP-1 and the well-characterized splice variant of RASSF1, RASSF1C, in cancer cell lines and primary tumors. We confirmed that the RASSF1A promoter is robustly methylated within a 32-CpG region in solid tumors and results in lower mRNA expression. The MOAP-1 promoter contains ~110 CpG sites, but was not found to be methylated in cancer cell lines when 19 predicted CpG sites were explored. Interestingly, MOAP-1 mRNA expression positively correlated with RASSF1A expression in numerous cancers, whereas RASSF1C expression remained the same or was increased in cell lines or tissues with epigenetic loss of RASSF1A. We speculate that MOAP-1 and RASSF1A may be more intimately connected than originally thought, and the expression of both are warranted in experimental designs exploring the biology of the RASSF1A/MOAP-1 molecular pathway.

Stress has long been thought of to be associated with increased risk of cancer. Chronic stress is associated with elevated levels of sympathetic neurotransmitter (norepinephrine and neuropeptide Y: NPY) release and immunosuppression. The expression of NPY receptors has been reported in human breast carcinomas. Recently, activation of the NPY Y5 receptor was shown to stimulate cell growth and increase migration in human breast cancer cells; however the effects of NPY have yet to be investigated in a murine model of breast cancer. Thus, the specific aims of the current study were to: (i) characterize NPY receptor expression in 4T1 breast cancer cells and orthotopic tumors grown in BALB/c mice and (ii) investigate the impact of NPY receptor activation on 4T1 cell proliferation and migration in vitro. Positive expression of NPY receptors (Y1R, Y2R and Y5R) was observed in cells and tumor tissue. As well, NPY treatment of 4T1 cells promoted a concentration-dependent increase in proliferation, through increased phosphorylation of ERK 1/2. Using NPY receptor antagonists (Y1R:BIBP3226, Y2R:BIIE0246 and Y5R:L-152,804), we found the proliferative response to be Y5R mediated. Additionally, NPY increased chemotaxis through Y2R and Y5R activation. These data are in congruence with those from human cell lines and highlight the 4T1 cell line as a translatable model of breast cancer in which the effects of NPY can be studied in an immunocompetent system.

ObjectivePolymerase epsilon (POLE) is a DNA polymerase with a proofreading (exonuclease) domain, responsible for the recognition and excision of mispaired bases, thereby allowing high-fidelity DNA replication to occur. The Cancer Genome Atlas research network recently identified an ultramutated group of endometrial carcinomas, characterized by mutations in POLE, and exceptionally high substitution mutation rates. These POLE mutated endometrial tumors were almost exclusively of the endometrioid histotype. The prevalence and patterns of POLE mutated tumors in endometrioid carcinomas of the ovary, however, have not been studied in detail.Materials and MethodsIn this study, we investigate the frequency of POLE exonuclease domain mutations in a series of 89 ovarian endometrioid carcinomas.ResultsWe found POLE mutations in 4 of 89 (4.5%) cases, occurring in 3 of 23 (13%) International Federation of Gynecology and Obstetrics (FIGO) grade 1, 1 of 43 (2%) FIGO grade 2, and 0 of 23 (0%) FIGO grade 3 tumors. All mutations were somatic missense point mutations, occurring at the commonly reported hotspots, P286R and V411L. All 3 POLE-mutated FIGO grade 1 tumors displayed prototypical histology, and the POLE-mutated FIGO grade 2 tumor displayed morphologic heterogeneity with focally high-grade features. All 4 patients with POLE-mutated tumors followed an uneventful clinical course with no disease recurrence; however, this finding was not statistically significant (P= 0.59).ConclusionsThe low rate of POLE mutations in ovarian endometrioid carcinoma and their predominance within the low FIGO grade tumors are in contrast to the findings in the endometrium.

Human embryonic stem cells (hESCs) offer exciting potential in regenerative medicine for the treatment of a host of diseases including cancer, Alzheimer's and Parkinson's disease. They also provide insight into human development and disease and can be used as models for drug discovery and toxicity analyses. The key properties of hESCs that make them so promising for medical use are that they have the ability to self-renew indefinitely in culture and they are pluripotent, which means that they can differentiate into any of more than 200 human cell types. Since proteins are the effectors of cellular processes, it is important to investigate hESC expression at the protein level as well as at the transcript level. In addition, post-translational modifications, such as phosphorylation, may influence the activity of pivotal proteins in hESCs, and this information can only be determined by studying the proteome. In this review, we summarize the results obtained from several proteomics analyses of hESCs that have been reported in the last few years.

Numerous matrices for the growth of human embryonic stem cells (hESC) in vitro have been described. However, their exact composition is typically unknown. Information on the components of these matrices will aid in the development of a fully defined growth surface for hESCs. These matrices typically consist of mixture of proteins present in a wide range of abundance making their characterization challenging. In this study, we performed the proteomic analysis of five previously uncharacterized matrices: CellStart, Human Basement Membrane Extract (Human BME), StemXVivo, Bridge Human Extracellular Matrix (BridgeECM), and mouse embryonic fibroblast conditioned matrix (MEF-CMTX). Based on a proteomics protocol optimized using lysates from HeLa cells, we undertook the analysis of the five complex extracellular matrix (ECM) samples using a combination of strong anion and cation exchange chromatography and SDS-PAGE. For each of these matrices, we identify numerous proteins, indicating their complex nature. We also compared these results with a similar proteomics analysis of the growth matrix, Matrigel™. From these analyses, we observed that fibronectin is a primary component of nearly all hESC supportive matrices. This observation led to the investigation of the suitability of fibronectin as a defined ECM for the growth of hESCs. We found that fibronectin promotes the maintenance of pluripotent H9 and CA1 hESCs in an undifferentiated state using mTeSR1 medium. This finding validates the utility of characterizing matrices used for hESC growth in revealing ECM components required for culturing hESCs in a universally applicable defined system.

Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself.