Jill Bayliss

Reversal of the Estrogen Receptor–Negative Phenotype in Breast Cancer and Restoration of Antiestrogen Response

Purpose: In breast cancer, the presence of estrogen receptor α (ER) denotes a better prognosis and response to antiestrogen therapy. Lack of ERα correlates with overexpression of epidermal growth factor receptor or c-erbB-2. We have shown that hyperactivation of mitogen-activated protein kinase (MAPK) directly represses ERα expression in a reversible manner. In this study, we determine if inhibition of MAPK in established ERα− breast cancer cell lines and tumors results in reexpression of ERα, and further, if reexpression of ERα in these ERα− tumors and cell lines could restore antiestrogen responses. Experimental Design: Established ERα− breast cancer cell lines, ERα− breast tumors, and tumor cell cultures obtained from ERα− tumors were used in this study. Inhibition of hyperactive MAPK was accomplished via the MAPK/ERK kinase 1/2 inhibitor U0126 or via upstream inhibition with Iressa or Herceptin. Western blotting or reverse transcription-PCR for ERα was used to assess the reexpression of ERα in cells treated with U0126. Growth assays with WST-1 were done to assess restoration of antiestrogen sensitivity in these cells. Results: Inhibition of MAPK activity in ERα− breast cancer cell lines results in reexpression of ERα; upstream inhibition via targeting epidermal growth factor receptor or c-erbB-2 is equally effective. Importantly, this reexpressed ERα can now mediate an antiestrogen response in a subset of these ERα− breast cancer cell lines. Treatment of ERα− tumor specimens with MAPK inhibitors results in restoration of ERα mRNA, and similarly in epithelial cultures from ERα− tumors, MAPK inhibition restores both ERα protein and antiestrogen response. Conclusions: These data show both the possibility of restoring ERα expression and antiestrogen responses in ERα− breast cancer and suggest that there exist ERα− breast cancer patients who would benefit from a combined MAPK inhibition/hormonal therapy.

Optimized polymeric film-based nitric oxide delivery inhibits bacterial growth in a mouse burn wound model.

Nitric oxide (NO) has many biological roles (e.g. antimicrobial agent, promoter of angiogenesis, prevention of platelet activation) that make NO releasing materials desirable for a variety of biomedical applications. Localized NO release can be achieved from biomedical grade polymers doped with diazeniumdiolated dibutylhexanediamine (DBHD/N2O2) and poly(lactic-co-glycolic acid) (PLGA). In this study, the optimization of this chemistry to create film/patches that can be used to decrease microbial infection at wound sites is examined. Two polyurethanes with different water uptakes (Tecoflex SG-80A (6.2±0.7wt.%) and Tecophilic SP-60D-20 (22.5±1.1wt.%)) were doped with 25wt.% DBHD/N2O2 and 10wt.% of PLGA with various hydrolysis rates. Films prepared with the polymer that has the higher water uptake (SP-60D-20) were found to have higher NO release and for a longer duration than the polyurethane with the lower water uptake (SG-80A). The more hydrophilic polymer enhances the hydrolysis rate of the PLGA additive, thereby providing a more acidic environment that increases the rate of NO release from the NO donor. The optimal NO releasing and control SG-80A patches were then applied to scald burn wounds that were infected with Acinetobacter baumannii. The NO released from these patches applied to the wounds is shown to significantly reduce the A. baumannii infection after 24h (∼4 log reduction). The NO release patches are also able to reduce the level of transforming growth factor-β in comparison to controls, which can enhance re-epithelialization, decrease scarring and reduce migration of bacteria. The combined DBHD/N2O2 and PLGA-doped polymer patches, which could be replaced periodically throughout the wound healing process, demonstrate the potential to reduce risk of bacterial infection and promote the overall wound healing process.

Lipopolysaccharide binding protein inhibitory peptide protects against acetaminophen-induced hepatotoxicity

Acetaminophen (APAP)-induced liver injury remains the main cause of acute liver failure in the United States. Our previous work demonstrated that LPS binding protein (LBP) knockout mice are protected from APAP-induced hepatotoxicity. LBP is known to bind avidly to LPS, facilitating cellular activation. In this study, we sought to specifically inhibit the interaction between LBP and LPS to define the role of this interaction in APAP-induced liver injury. The peptide LBPK95A was able to inhibit LBP-mediated LPS activation of RAW 267.4 cells in a dose-dependent manner in vitro. In vivo, C57Bl/6 mice were treated with either LBPK95A or vehicle control concurrently with the administration of APAP (350 mg/kg). Mice treated with LBPK95A had significantly lower serum aspartate aminotransferase and alanine aminotransferase levels. Morphometric analysis of the liver tissue showed significantly less liver injury in mice treated with LBPK95A. To assess whether the LBPK95A altered glutathione depletion and APAP metabolism, we measured total glutathione levels in the liver after APAP. We found no difference in the glutathione levels and APAP-adduct formation between LBPK95A vs. vehicle control both at baseline and after APAP. In conclusion, our results support the hypothesis that LBP-induced liver injury after APAP is due to its ability to mediate activation by endogenous LPS. Our results suggest that blocking LBP-LPS interactions is a potential therapeutic avenue for the treatment of APAP-induced liver injury.

Lowered H3K27me3 and DNA hypomethylation define poorly prognostic pediatric posterior fossa ependymomas

A subset of childhood posterior fossa ependymomas with poor prognosis is epigenetically similar to H3K27M gliomas. Epigenetics helps find the bad tumors Ependymomas are brain tumors that can occur in people of all ages and in different parts of the central nervous system. The prognosis of these tumors does not necessarily correlate with clinical characteristics or even tumor grade, and there are no recurrent genetic mutations that can be used to classify these tumors. To address this problem, Bayliss et al. examined the epigenetics of ependymoma. By doing this, the authors identified some characteristic methylation patterns that correlate with prognosis, including one specific pattern that is also seen in childhood gliomas and associated with more invasive tumors. Childhood posterior fossa (PF) ependymomas cause substantial morbidity and mortality. These tumors lack recurrent genetic mutations, but a subset of these ependymomas exhibits CpG island (CpGi) hypermethylation [PF group A (PFA)], implicating epigenetic alterations in their pathogenesis. Further, histological grade does not reliably predict prognosis, highlighting the importance of developing more robust prognostic markers. We discovered global H3K27me3 reduction in a subset of these tumors (PF-ve ependymomas) analogous to H3K27M mutant gliomas. PF-ve tumors exhibited many clinical and biological similarities with PFA ependymomas. Genomic H3K27me3 distribution showed an inverse relationship with CpGi methylation, suggesting that CpGi hypermethylation drives low H3K27me3 in PF-ve ependymomas. Despite CpGi hypermethylation and global H3K27me3 reduction, these tumors showed DNA hypomethylation in the rest of the genome and exhibited increased H3K27me3 genomic enrichment at limited genomic loci similar to H3K27M mutant gliomas. Combined integrative analysis of PF-ve ependymomas with H3K27M gliomas uncovered common epigenetic deregulation of select factors that control radial glial biology, and PF radial glia in early human development exhibited reduced H3K27me3. Finally, H3K27me3 immunostaining served as a biomarker of poor prognosis and delineated radiologically invasive tumors, suggesting that reduced H3K27me3 may be a prognostic indicator in PF ependymomas.

Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice.

Extracellular adenosine triphosphate (ATP), present in thermally injured tissue, modulates the inflammatory response and causes significant tissue damage. The authors hypothesize that neutrophil infiltration and ensuing tissue necrosis would be mitigated by removing ATP-dependent signaling at the burn site. Mice were subjected to 30% TBSA partial-thickness scald burn by dorsal skin immersion in a water bath at 60 or 20°C (nonburn controls). In the treatment arm, an ATP hydrolyzing enzyme, apyrase, was applied directly to the site immediately after injury. Skin was harvested after 24 hours and 5 days for hematoxylin and eosin stain, elastase, and Ki-67 staining. Tumor necrosis factor (TNF)-&agr; and interferon (IFN)-&bgr; expression were measured through quantitative real-time polymerase chain reaction. At 24 hours, the amount of neutrophil infiltration was different between the burn and burn + apyrase groups (P < .001). Necrosis was less extensive in the apyrase group when compared with the burn group at 24 hours and 5 days. TNF-&agr; and IFN-&bgr; expression at 24 hours in the apyrase group was lower than in the burn group (P < .05). However, Ki-67 signaling was not significantly different among the groups. The results of this study support the role of extracellular ATP in neutrophil activity. The authors demonstrate that ATP hydrolysis at the burn site allays the neutrophil response to thermal injury and reduces tissue necrosis. This decrease in inflammation and tissue necrosis is at least partially because of TNF-&agr; and IFN-&bgr; signaling. Apyrase could be used as topical inflammatory regulators to quell the injury caused by inflammation.

Lipopolysaccharide binding protein inhibitory peptide alters hepatic inflammatory response post-hemorrhagic shock

Translocation of microorganisms and endotoxin (LPS) across the gastrointestinal mucosa may exacerbate the inflammatory response and potentiate hepatic injury associated with hemorrhagic shock. Lipopolysaccharide binding protein (LBP) augments LPS signaling through TLR4. In addition, evidence suggests that TLR4-mediated injury in liver ischemia/reperfusion occurs through the IRF-3/MyD88 independent pathway. We hypothesized that administration of LBP inhibiting peptide, LBPK95A, given at the time of resuscitation would reduce liver inflammation and injury in a murine model of hemorrhagic shock by limiting LPS-induced activation of the MyD88 independent pathway. Hemorrhagic shock was induced in male, C57BL/6 mice; a mean arterial blood pressure of 35 mmHg was maintained for 2.5 h. LBPK95A peptide or equal volume Lactated Ringer’s solution was administered followed by fluid resuscitation. Mice were sacrificed at 2 and 6 h post-resuscitation. At 2 h, liver mRNA levels revealed a significant reduction in IFN-β, a cytokine produced via the MyD88 independent pathway, with LBPK95A treatment. However, mRNA levels of TNF-α, a cytokine associated with the MyD88 dependent pathway, were unaffected by treatment. The LBP inhibitory peptide did selectively reduce activation of TLR4 signaling via the IRF-3/MyD88 independent pathway. These results suggest that LBP promotes cytokine production through the MyD88 independent pathway during hemorrhagic shock.

Abstract 3863: A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas

Pediatric posterior fossa ependymomas are poorly understood childhood brain tumors and have no effective treatments. The biology of these tumors is obscure as recent sequencing efforts suggest that they lack recurrent genetic alterations. A subset of these tumors termed PF-A ependymomas exhibits CpG-island hypermethylation implicating epigenetic alterations in their pathogenesis. Through comprehensive analyses of histone modification, we discovered global H3K27me3 reduction in a subset of these tumors. Tumors with lowered H3K27me3 showed many clinical and biologic similarities with PFA-ependymomas. Global reduction in H3K27me3 is likewise observed in pediatric gliomas that bear histone H3K27M mutations termed diffuse intrinsic pontine gliomas (DIPG) that also arise in the posterior fossa of young children. Analyses of ependymomas with reduced H3K27me3 and H3K27M mutant DIPGs showed many similarities in DNA methylation and enrichment of H3K27me3 in many genomic loci important for neuroglial specification. Combined integrative analysis of both tumor types uncovered common epigenetic deregulation of select factors that control radial glial biology and radial glia in the developing posterior fossa showed reduced H3K27me3. Finally, PF ependymomas with lowered H3K27me3 were more invasive radiologically and exhibited poor prognosis in three independent cohorts (P 300). These data have clinical implications for biomarker development and to inform epigenetic approaches to treat PF ependymomas. Citation Format: Sriram Venneti, Jill Bayliss, Piali Mukherjee, Chao Lu, Siddhant Jain, Chan Chung, Daniel Martinez, Benjamin Sabari, Ashley Margol, Pooja Panwalkar, Abhijit Paroloia, Melike Pekmezci, Richard Mc Eachin, Marcin Cieslik, Benita Tamrazi, Benjamin Garcia, Gaspare La Rocca, Mariarita Santi, Peter Lewis, Cynthia Hawkins, Ari Melnick, C David Allis, Craig B. Thompson, Arul Chinnaiyan, Alexander R. Judkins. A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas [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 3863. doi:10.1158/1538-7445.AM2017-3863

Apyrase elicits host antimicrobial responses and resolves infection in burns

The authors previously reported that adenosine triphosphate (ATP) stimulates biofilm formation and removal of the ATP could reduce biofilm formation. The main objective of this study was to evaluate the effects of the ATP-hydrolyzing enzyme, apyrase, on control of Acinetabacter baumannii infection in the burn wound as well as to assess host skin antimicrobial responses. The authors found that apyrase stimulated nitric oxide formation at the wound site and reduced CD55 expression, thereby inducing the assembly of membrane attack complexes. Apyrase treatment nearly eradicated multidrug-resistant A. baumannii from burn wounds in the absence of antibiotics. Apyrase may be an effective therapy against antibiotic-resistant bacterial infections in burns.

The re-expression of estrogen receptor in estrogen receptor-negative breast cancer and restoration of anti-estrogen responses

Loss of estrogen receptor α ERα in breast cancer correlates with a more aggressive, tamoxifen resistant phenotype. ERα-negative tumors often display overexpression or amplification of growth factor receptors of the erbB family, particularly EGFR and erbB-2, and consequently, elevated growth factor signaling and resultant MAP kinase (ERK) activity. We have previously shown that overexpression/hyperactivation of EGFR or erbB-2, or the downstream effectors Raf or MEK, in ERα+, estrogen-dependent MCF-7 cells results in the acquisition of estrogen-independence and loss of ERα expression. We have shown that the common downstream effector of ERα downregulation in all our model cell lines is hyperactive MAPK and that inhibition of this hyperactive MAPK restores ERα expression. Microarray expression profiling of these hyperactive MAPK model cell lines revealed a hyperactive MAPK signature that correlates with ERα-breast cancer and not ERα+ breast cancer. We have more recently extended these observations to established ERα-breast cancer cell lines and primary cultures from ERα-breast tumor specimens. Inhibition of MAPK in these ERα-breast cancer cells restores ERα expression and associated with this re-expression of ERα is the acquisition of anti-estrogen responses. These data demonstrate the dynamic nature of ERα expression in breast cancer cells and the ability to impact ERα expression by altering cellular signaling pathways. Further, they suggest a potential novel therapeutic strategy for ERα-breast cancer: inhibition of MAPK activity to restore both ERα expression and anti-estrogen responses.