Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. METHODS: We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. RESULTS: We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA-protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. CONCLUSIONS: This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Miller (Genee-Wiedemann) syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH.

Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase (DHODH)], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genée-Weidemann or postaxial acrofacial dysostosis) syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes (CAD, UMPS), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh, Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.

Host obesity alters the ovarian tumor immune microenvironment and impacts response to standard of care chemotherapy.

BACKGROUND: The majority of women with epithelial ovarian cancer (OvCa) are diagnosed with metastatic disease, resulting in a poor 5-year survival of 31%. Obesity is a recognized non-infectious pandemic that increases OvCa incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity mouse model wherein a significantly enhanced tumor burden was associated with a decreased M1/M2 tumor-associated macrophage ratio (Liu Y et al. Can, Res. 2015; 75:5046-57). METHODS: The objective of this study was to use pre-clinical murine models of diet-induced obesity to evaluate the effect of a high fat diet (HFD) on response to standard of care chemotherapy and to assess obesity-associated changes in the tumor microenvironment. Archived tumor tissues from ovarian cancer patients of defined body mass index (BMI) were also evaluated using multiplexed immunofluorescence analysis of immune markers. RESULTS: We observed a significantly diminished response to standard of care paclitaxel/carboplatin chemotherapy in HFD mice relative to low fat diet (LFD) controls. A corresponding decrease in the M1/M2 macrophage ratio and enhanced tumor fibrosis were observed both in murine DIO studies and in human tumors from women with BMI > 30. CONCLUSIONS: Our data suggest that the reported negative impact of obesity on OvCa patient survival may be due in part to the effect of the altered M1/M2 tumor-associated macrophage ratio and enhanced fibrosis on chemosensitivity. These data demonstrate a contribution of host obesity to ovarian tumor progression and therapeutic response and support future combination strategies targeting macrophage polarization and/or fibrosis in the obese host.

Intracerebral lipopolysaccharide induces neuroinflammatory change and augmented brain injury in growth-restricted neonatal rats.

INTRODUCTION: Intrauterine growth restriction (IUGR) alters fetal development and is associated with neurodevelopmental abnormalities. We hypothesized that growth restriction from reduced intrauterine perfusion would predispose neonatal rats to subsequent inflammatory brain injury. METHODS: In this study, IUGR was achieved by induced placental insufficiency in pregnant rats at 14 days of gestation. IUGR offspring and sham-operated control pups were subsequently injected with intracerebral lipopolysaccharide (LPS) as a model of periventricular leukomalacia (PVL). RESULTS: LPS similarly elevates proinflammatory cytokines in the brains of both IUGR and control rat pups. However, the chemokines cytokine-induced neutrophil chemoattractant-1 (CINC-1) and macrophage chemoattractant protein-1 (MCP-1), as well as microglia activation, were significantly higher in LPS-treated IUGR rat pups as compared with LPS-treated controls. In addition to the unique brain inflammatory response, IUGR rat pups demonstrated increased brain damage with an increased number of apoptotic cells, larger lateral ventricular size, and more severe impairment of myelination. DISCUSSION: This study provides evidence that placental insufficiency may sensitize the innate immune system in the immature brain and reveals a possible link between brain inflammation and injury.

Dexamethasone and betamethasone protect against lipopolysaccharide-induced brain damage in neonatal rats.

INTRODUCTION: The aim of this study was to test whether dexamethasone (Dex) and betamethasone (Beta), two of the most commonly used corticosteroids, protect against lipopolysaccharide (LPS)-induced white matter damage and neurobehavioral dysfunction. METHODS: LPS or sterile saline was injected into the brain white matter of rat pups at postnatal day 5 (P5), and Dex or Beta was given intraperitoneally to the rat pups 1 h before the LPS microinjection. Brain inflammatory response, brain damage, and myelination were examined at P6, P8, and P14. Neurobehavioral tests were performed from P3 through P22. RESULTS: Our results demonstrate that Dex and Beta markedly diminish the LPS-induced brain inflammatory response, restore myelin basic protein (MBP) expression, and alleviate lateral ventricle dilation. Both corticosteroids demonstrate significant protection against most LPS-induced behavioral deficits, including those in rearing, vibrissa-elicited forelimb-placing, beam walking, learning, and elevated plus-maze test. Of note, only Beta improved the locomotion and stereotype dysfunction. In contrast to their beneficial effects, neither drug prevented LPS-induced delay in body weight gain from P6 through P21. DISCUSSION: Our study suggests that if their adverse effects are minimized, corticosteroids may be the potential candidate drugs to prevent brain damage in premature infants.

Plasmids shape the diverse accessory resistomes of Escherichia coli ST131.

The human gut microbiome includes beneficial, commensal and pathogenic bacteria that possess antimicrobial resistance (AMR) genes and exchange these predominantly through conjugative plasmids. Escherichia coli is a significant component of the gastrointestinal microbiome and is typically non-pathogenic in this niche. In contrast, extra-intestinal pathogenic E. coli (ExPEC) including ST131 may occupy other environments like the urinary tract or bloodstream where they express genes enabling AMR and host cell adhesion like type 1 fimbriae. The extent to which commensal E. coli and uropathogenic ExPEC ST131 share AMR genes remains understudied at a genomic level, and we examined this here using a preterm infant resistome. We found that individual ST131 had small differences in AMR gene content relative to a larger shared resistome. Comparisons with a range of plasmids common in ST131 showed that AMR gene composition was driven by conjugation, recombination and mobile genetic elements. Plasmid pEK499 had extended regions in most ST131 Clade C isolates, and it had evidence of a co-evolutionary signal based on protein-level interactions with chromosomal gene products, as did pEK204 that had a type IV fimbrial pil operon. ST131 possessed extensive diversity of selective type 1, type IV, P and F17-like fimbriae genes that was highest in subclade C2. The structure and composition of AMR genes, plasmids and fimbriae vary widely in ST131 Clade C and this may mediate pathogenicity and infection outcomes.

In vivo selection of highly metastatic human ovarian cancer sublines reveals role for AMIGO2 in intra-peritoneal metastatic regulation.

The majority of women with ovarian cancer are diagnosed with metastatic disease, therefore elucidating molecular events that contribute to successful metastatic dissemination may identify additional targets for therapeutic intervention and thereby positively impact survival. Using two human high grade serous ovarian cancer cell lines with inactive TP53 and multiple rounds of serial in vivo passaging, we generated sublines with significantly accelerated intra-peritoneal (IP) growth. Comparative analysis of the parental and IP sublines identified a common panel of differentially expressed genes. The most highly differentially expressed gene, upregulated by 60-65-fold in IP-selected sublines, was the type I transmembrane protein AMIGO2. As the role of AMIGO2 in ovarian cancer metastasis remains unexplored, CRISPR/Cas9 was used to reduce AMIGO2 expression, followed by in vitro and in vivo functional analyses. Knockdown of AMIGO2 modified the sphere-forming potential of ovarian cancer cells, reduced adhesion and invasion in vitro, and significantly attenuated IP metastasis. These data highlight AMIGO2 as a new target for a novel anti-metastatic therapeutic approach aimed at blocking cohesion, survival, and adhesion of metastatic tumorspheres.

IGF-1 can either protect against or increase LPS-induced damage in the developing rat brain.

Periventricular leukomalacia (PVL) is a major form of brain damage in premature infants. This study was to test whether IGF-1 can prevent PVL-like brain damage induced by lipopolysaccharide (LPS) in the neonatal rat. Intraventricular delivery of LPS resulted in an acute brain inflammatory response, i.e., rapid recruitment of polymorphonuclear leukocytes (PMNs), activation of microglia and astrocytes, and induction of IL-1beta (IL1beta) expression. Brain inflammation was associated with the loss of O4+ preoligodendrocytes (preOLs), a decrease of myelin basic protein (MBP) in the white matter and an increase of pyknotic cells in the cortex. IGF-1 at a low dose significantly prevented LPS-induced deleterious effects without alteration of IL-1beta expression and microglia/astrocytes activation. On the other hand, the low dose of IGF-1 enhanced LPS-induced PMNs recruitment and blood-brain barrier (BBB) permeability, and caused intracerebral hemorrhage. At higher doses, co-application of IGF-1 with LPS resulted in a high mortality rate. Brains from the surviving rats showed massive PMN infiltration and intracerebral hemorrhage. However, these adverse effects were not found in rats treated with IGF-1 alone. This study provides the alarming evidence that in an acute inflammatory condition, IGF-1 may have severe, harmful effects on the developing brain.

Lysophosphatidic acid modulates ovarian cancer multicellular aggregate assembly and metastatic dissemination.

Epithelial ovarian cancer (EOC) metastasis occurs by exfoliation of cells and multicellular aggregates (MCAs) from the tumor into the peritoneal cavity, adhesion to and retraction of peritoneal mesothelial cells and subsequent anchoring. Elevated levels of lysophosphatidic acid (LPA) have been linked to aberrant cell proliferation, oncogenesis, and metastasis. LPA disrupts junctional integrity and epithelial cohesion in vitro however, the fate of free-floating cells/MCAs and the response of host peritoneal tissues to LPA remain unclear. EOC MCAs displayed significant LPA-induced changes in surface ultrastructure with the loss of cell surface protrusions and poor aggregation, resulting in increased dissemination of small clusters compared to untreated control MCAs. LPA also diminished the adhesive capacity of EOC single cells and MCAs to murine peritoneal explants and impaired MCA survival and mesothelial clearance competence. Peritoneal tissues from healthy mice injected with LPA exhibited enhanced mesothelial surface microvilli. Ultrastructural alterations were associated with restricted peritoneal susceptibility to metastatic colonization by single cells as well as epithelial-type MCAs. The functional consequence is an LPA-induced dissemination of small mesenchymal-type clusters, promoting a miliary mode of peritoneal seeding that complicates surgical removal and is associated with worse prognosis.

The awesome power of multiple model systems: interpreting the complex nature of spindle checkpoint signaling.

The spindle checkpoint coordinates the cell biology of mitosis with cell-cycle progression. It ensures that sister-chromatid separation only takes place when all kinetochores have formed stable bipolar microtubule attachments. Here, we discuss recent advances in our understanding of what activates this checkpoint pathway, the molecular nature of the checkpoint signal and its mode of transmission, and how the checkpoint might be inactivated.

Modeling the effect of ascites-induced compression on ovarian cancer multicellular aggregates.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. EOC dissemination is predominantly via direct extension of cells and multicellular aggregates (MCAs) into the peritoneal cavity, which adhere to and induce retraction of peritoneal mesothelium and proliferate in the submesothelial matrix to generate metastatic lesions. Metastasis is facilitated by the accumulation of malignant ascites (500 ml to >2 l), resulting in physical discomfort and abdominal distension, and leading to poor prognosis. Although intraperitoneal fluid pressure is normally subatmospheric, an average intraperitoneal pressure of 30 cmH2O (22.1 mmHg) has been reported in women with EOC. In this study, to enable experimental evaluation of the impact of high intraperitoneal pressure on EOC progression, two new in vitro model systems were developed. Initial experiments evaluated EOC MCAs in pressure vessels connected to an Instron to apply short-term compressive force. A Flexcell Compression Plus system was then used to enable longer-term compression of MCAs in custom-designed hydrogel carriers. Results show changes in the expression of genes related to epithelial-mesenchymal transition as well as altered dispersal of compressed MCAs on collagen gels. These new model systems have utility for future analyses of compression-induced mechanotransduction and the resulting impact on cellular responses related to intraperitoneal metastatic dissemination.This article has an associated First Person interview with the first authors of the paper.

Selenium and sulforaphane modify the expression of selenoenzymes in the human endothelial cell line EAhy926 and protect cells from oxidative damage.

OBJECTIVE: We examined the ability of sulforaphane and selenium to modify the expression of thioredoxin reductase (TR-1) and the glutathione peroxidases (GPX-1 and GPX-4) in EAhy926 cells. The effectiveness of these agents to protect cells against peroxidative damage was also assessed. METHODS: EAhy926 cells were supplemented with 40 nM of selenite and/or sulforaphane (10 microM) for 72 h and the expression of TR-1, GPX-1, and GPX-4 was assessed. Parallel cultures of selenium- and sulforaphane-treated cells were exposed to tertiary butyl hydroperoxide (t-BuOOH; 0-500 microM) for 20 h, and cell integrity was determined by the percentage of lactate dehydrogenase retained by the cellular layer. RESULTS: Selenite treatment increased the concentration of TR-1 (1.6 +/- 0.17 fold, P < 0.05), GPX-1 activity (8.2 +/- 1.08 fold, P < 0.001), and GPX-4 activity (3.1 +/- 0.25 fold, P < 0.001). Sulforaphane induced TR-1 expression in selenium-deficient cells (1.83 +/- 0.11 fold, P < 0.001) and selenium-supplemented cells (2.90 +/- 0.17 fold, P < 0.001) but had no inductive effect on GPX-1 or GPX-4. The combination of selenite and sulforaphane produced an increase in TR-1 expression that was significantly greater (P < 0.001) than that achieved when each agent was added alone. Selenium and sulforaphane acted in a synergistic manner to protect cells from damage caused by t-BuOOH. The susceptibility of cells to damage by t-BuOOH increased in this order: control > sulforaphane > selenite > selenite + sulforaphane (P < 0.0001). CONCLUSION: In endothelial cells, sulforaphane increases TR-1 but not GPX-1 and GPX-4 and in doing so confers protection against oxidative damage induced by lipid hydroperoxides. The results highlight the potential important role of TR-1 over the GPXs in protecting endothelial cells from oxidative cell damage. We also suggest that our results indicate a potential beneficial role for sulforaphane in protecting the vascular endothelium from oxidative damage.

Heterogeneous Cadherin Expression and Multicellular Aggregate Dynamics in Ovarian Cancer Dissemination.

Epithelial ovarian carcinoma spreads via shedding of cells and multicellular aggregates (MCAs) from the primary tumor into peritoneal cavity, with subsequent intraperitoneal tumor cell:mesothelial cell adhesion as a key early event in metastatic seeding. Evaluation of human tumor extracts and tissues confirms that well-differentiated ovarian tumors express abundant E-cadherin (Ecad), whereas advanced lesions exhibit upregulated N-cadherin (Ncad). Two expression patterns are observed: "mixed cadherin," in which distinct cells within the same tumor express either E- or Ncad, and "hybrid cadherin," wherein single tumor cell(s) simultaneously expresses both cadherins. We demonstrate striking cadherin-dependent differences in cell-cell interactions, MCA formation, and aggregate ultrastructure. Mesenchymal-type Ncad+ cells formed stable, highly cohesive solid spheroids, whereas Ecad+ epithelial-type cells generated loosely adhesive cell clusters covered by uniform microvilli. Generation of "mixed cadherin" MCAs using fluorescently tagged cell populations revealed preferential sorting into cadherin-dependent clusters, whereas mixing of cell lines with common cadherin profiles generated homogeneous aggregates. Recapitulation of the "hybrid cadherin" Ecad+/Ncad+ phenotype, via insertion of the CDH2 gene into Ecad+ cells, resulted in the ability to form heterogeneous clusters with Ncad+ cells, significantly enhanced adhesion to organotypic mesomimetic cultures and peritoneal explants, and increased both migration and matrix invasion. Alternatively, insertion of CDH1 gene into Ncad+ cells greatly reduced cell-to-collagen, cell-to-mesothelium, and cell-to-peritoneum adhesion. Acquisition of the hybrid cadherin phenotype resulted in altered MCA surface morphology with increased surface projections and increased cell proliferation. Overall, these findings support the hypothesis that MCA cadherin composition impacts intraperitoneal cell and MCA dynamics and thereby affects ultimate metastatic success.

Analysis of Bub3 spindle checkpoint function in Xenopus egg extracts.

The spindle checkpoint delays the onset of anaphase if there are any defects in the interactions between spindle microtubules and kinetochores. This checkpoint has been reconstituted in vitro in Xenopus egg extracts, and here we use antibodies to Xenopus Bub3 (XBub3) to show that this protein is required for both the activation and the maintenance of a spindle checkpoint arrest in egg extracts. We detect two forms of XBub3 in egg extracts and find both to be complexed with the XBub1 and XBubR1 kinases. Only one form of XBub3 is apparent in Xenopus tissue culture (XTC) cells, and localisation studies reveal that, unlike the Mad proteins, which are concentrated at the nuclear periphery, XBub3 is diffusely localised throughout the nucleus during interphase. During early prophase it is recruited to kinetochores, where it remains until chromosomes align at the metaphase plate. We discuss the mechanism by which our alpha-XBub3 antibodies interfere with the checkpoint and possible roles for XBub3 in the spindle checkpoint pathway.