Search

Tumor sample viability

Disease models, including in vitro cell culture and animal models, have contributed significantly to developing diagnostics and treatments over the past several decades. The successes of traditional drug screening methods were generally hampered by not adequately mimicking critical in vivo features, such as a 3D microenvironment and dynamic drug diffusion through the extracellular matrix (ECM). To address these issues, we developed a 3D dynamic drug delivery system for cancer drug screening that mimicks drug dissemination through the tumor vasculature and the ECM by creating collagen-embedded microfluidic channels. Using this novel 3D ECM microsystem, we compared viability of tumor pieces with traditionally used 2D methods in response to three different drug combinations. Drug diffusion profiles were evaluated by simulation methods and tested in the 3D ECM microsystem and a 2D 96-well setup. Compared with the 2D control, the 3D ECM microsystem produced reliable data on viability, drug ratios, and combination indeces. This novel approach enables higher throughput and sets the stage for future applications utilizing drug sensitivity predicting algorithms based on dynamic diffusion profiles requiring only minimal patient tissue. Our findings moved drug sensitivity screening closer to clinical implications with a focus on testing combinatorial drug effects, an option often limited by the amount of available patient tissues.

Autophagy is activated in response to cellular stressors and mediates lysosomal degradation and recycling of cytoplasmic material and organelles as a temporary cell survival mechanism. Defective autophagy is implicated in human pathology, as disruption of protein and organelle homeostasis enables disease-promoting mechanisms such as toxic protein aggregation, oxidative stress, genomic damage, and inflammation. We previously showed that autophagy-defective immortalized mouse mammary epithelial cells are susceptible to metabolic stress, DNA damage, and genomic instability. We now report that autophagy deficiency is associated with endoplasmic reticulum (ER) and oxidative stress, and with deregulation of p62-mediated keratin homeostasis in mammary cells, allograft tumors, and mammary tissues from genetically engineered mice. In human breast tumors, high phospho(Ser73)-K8 levels are inversely correlated with Beclin 1 expression. Thus, autophagy preserves cellular fitness by limiting ER and oxidative stress, a function potentially important in autophagy-mediated suppression of mammary tumorigenesis. Furthermore, autophagy regulates keratin homeostasis in the mammary gland via a p62-dependent mechanism. High phospho(Ser73)-K8 expression may be a marker of autophagy functional status in breast tumors and, as such, could have therapeutic implications for breast cancer patients. Mol Cancer Res; 8(6); 873–84. ©2010 AACR.

Supplementary Figures S1-S2; Supplementary Materials and Methods; Supplementary Table S1.

Out of all breast cancers (BC), ~20% are triple negative breast cancers (TNBC) devoid of the three receptors that define breast cancer treatment strategies such as estrogen receptor (ER), progesterone receptor (PR) and ERBB2 (also known as HER2). Unlike other BC, TNBC disproportionally affects younger women and those of African origins and recurs early within 5 years after diagnosis in ~ 40% of the patients. Standard care for TNBC patients includes surgery, ionizing radiation (IR) treatment and chemotherapy. Successful targeting of the poly (ADP-ribose) polymerase (PARP) has revolutionized therapy for cancer patients carrying gmBRCA1/2. However, in TNBC patients optimal clinical usage of PARP inhibitors (PARPi) is stifled by a) only 15-20% of TNBC patients being positive for gmBRCA1/2 and currently receiving PARPi therapy, b) most PARPi-treated gmBRCA1/2 patients developing resistance due to restoration of HR-mediated DNA DSB repair and, c) PARPi treatment-associated toxicities that require dose adaptations or drug discontinuation. Our novel therapeutic approach addresses this clinical unmet need by further advancing a small molecule inhibitor of HR that includes the 80% TNBC patients wild type for BRCA1/2 genes, re-sensitizes patients resistant to PARPi due to restoration of HR-mediated DNA DSB repair and lowers PARPi doses by conferring greater sensitivity to PARPi actions through synthetic lethality approaches.We first defined OA-NO2 ((E)-9/10-nitro-octadec-9-enoic acid), an endogenous electrophilic fatty acid nitroalkene as an RAD51 inhibitor in TNBC. RAD51 is essential in HR-mediated DNA DSB repair, is a highly coveted drug target in cancer and is a revealing functional biomarker for PARPi sensitivity. OA-NO2 reacts with protein thiols via reverse Michael addition reaction and has broad spectrum anticancer activities. OA-NO2 inhibits RAD51 function by selectively alkylating RAD51 on Cys319, a residue essential in RAD51- guided HR. In combination with the PARPi talazoparib, OA-NO2 synergizes the killing of TNBC cells in vitro and in vivo. Two reasons motivated the further development of OA-NO2 as RAD51 inhibitor: 1) X-ray crystal structure modeling predicted superior fitting with the RAD51 C-terminus if the nitroalkene substituent is closer to the carboxylate terminus of the nitro-fatty acid, and 2) OA-NO2 exhibits off target effects by inhibiting c-GAS-STING signaling and activating Nrf2 activity, thus, possibly offsetting anti-cancer activities. To optimize nitroalkene-mediated RAD51 inhibition, a library of >50 compounds was built that expands upon 5 chemical classes. The library was evaluated for structure activity relationships (SAR) and a new lead nitroalkene emerged, CP-23. This electrophilic small molecule kills as a monotherapy TNBC and not benign breast epithelial cells and unlike OA-NO2, has no impact on STING and Nrf2 signaling, or the cell cycle. CP-23 inhibits RAD51 DNA binding, shows potent inhibition of RAD51 nuclear foci formation after ionizing radiation (IR) treatment and HR in a GFP-reporter cell line, and demonstrated drug synergism with PARP inhibition and IR. At present, in vivo tumor growth inhibition, expansion of PARPi-CP-23 synthetic lethality studies and pilot PK/toxicology evaluation are underway. Citation Format: Carola Anke Neumann, John J Skoko, Bruce A Freeman, Francisco Schopfer, Steven Robert Woodcock, Fei Chang, Lisa Hong, Dennis Carl Braden, Crystal Uvalle. Targeting homologous recombination-proficient triple negative cancer cells with a novel RAD51 inhibitor [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-17.

BACKGROUND: Peroxiredoxin 1 (PRDX1) belongs to an abundant family of peroxidases whose role in cancer is still unresolved. While mouse knockout studies demonstrate a tumour suppressive role for PRDX1, in cancer cell xenografts, results denote PRDX1 as a drug target. Probably, this phenotypic discrepancy stems from distinct roles of PRDX1 in certain cell types or stages of tumour progression. METHODS: We demonstrate an important cell-autonomous function for PRDX1 utilising a syngeneic mouse model (BALB/c) and mammary fibroblasts (MFs) obtained from it. RESULTS: Loss of PRDX1 in vivo promotes collagen remodelling known to promote breast cancer progression. PRDX1 inactivation in MFs occurs via SRC-induced phosphorylation of PRDX1 TYR194 and not through the expected direct oxidation of CYS52 in PRDX1 by ROS. TYR194-phosphorylated PRDX1 fails to bind to lysyl oxidases (LOX) and leads to the accumulation of extracellular LOX proteins which supports enhanced collagen remodelling associated with breast cancer progression. CONCLUSIONS: This study reveals a cell type-specific tumour suppressive role for PRDX1 that is supported by survival analyses, depending on PRDX1 protein levels in breast cancer cohorts.

Homologous recombination (HR)-directed DNA double-strand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO(2)) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and γ-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO(2) inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO(2) inhibits HR and not nonhomologous end joining (NHEJ). OA-NO(2) alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO(2) because nonnitrated and saturated nonelectrophilic analogs of OA-NO(2), octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO(2) alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO(2)–mediated Cys-319 alkylation on ABL1 binding and found that OA-NO(2) inhibits RAD51–ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51–ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO(2) and suggests further investigation of lipid electrophile–based combinational therapies for TNBC.

Disease models, including in vitro cell culture and animal models, have contributed significantly to developing diagnostics and treatments over the past several decades. The successes of traditional drug screening methods were generally hampered by not adequately mimicking critical in vivo features, such as a 3D microenvironment and dynamic drug diffusion through the extracellular matrix (ECM). To address these issues, we developed a 3D dynamic drug delivery system for cancer drug screening that mimicks drug dissemination through the tumor vasculature and the ECM by creating collagen-embedded microfluidic channels. Using this novel 3D ECM microsystem, we compared viability of tumor pieces with traditionally used 2D methods in response to three different drug combinations. Drug diffusion profiles were evaluated by simulation methods and tested in the 3D ECM microsystem and a 2D 96-well setup. Compared with the 2D control, the 3D ECM microsystem produced reliable data on viability, drug ratios, and combination indeces. This novel approach enables higher throughput and sets the stage for future applications utilizing drug sensitivity predicting algorithms based on dynamic diffusion profiles requiring only minimal patient tissue. Our findings moved drug sensitivity screening closer to clinical implications with a focus on testing combinatorial drug effects, an option often limited by the amount of available patient tissues.

SIGNIFICANCE: It has been proposed that cancer cells are heavily dependent on their antioxidant defenses for survival and growth. Peroxiredoxins are a family of abundant thiol-dependent peroxidases that break down hydrogen peroxide, and they have a central role in the maintenance and response of cells to alterations in redox homeostasis. As such, they are potential targets for disrupting tumor growth. RECENT ADVANCES: Genetic disruption of peroxiredoxin expression in mice leads to an increased incidence of neoplastic disease, consistent with a role for peroxiredoxins in protecting genomic integrity. In contrast, many human tumors display increased levels of peroxiredoxin expression, suggesting that strengthened antioxidant defenses provide a survival advantage for tumor progression. Peroxiredoxin inhibitors are being developed and explored as therapeutic agents in different cancer models. CRITICAL ISSUES: It is important to complement peroxiredoxin knockout and expression studies with an improved understanding of the biological function of the peroxiredoxins. Although current results can be interpreted within the context that peroxiredoxins scavenge hydroperoxides, some peroxiredoxin family members appear to have more complex roles in regulating the response of cells to oxidative stress through protein interactions with constituents of other signaling pathways. FUTURE DIRECTIONS: Further mechanistic information is required for understanding the role of oxidative stress in cancer, the function of peroxiredoxins in normal versus cancer cells, and for the design and testing of specific peroxiredoxin inhibitors that display selectivity to malignant cells. Antioxid. Redox Signal. 28, 591–608.

Triple-negative breast cancer (TNBC) comprises ∼20% of all breast cancers and is the most aggressive mammary cancer subtype. Devoid of the estrogen and progesterone receptors, along with the receptor tyrosine kinase ERB2 (HER2), that define most mammary cancers, there are no targeted therapies for patients with TNBC. This, combined with a high metastatic rate and a lower 5-year survival rate than for other breast cancer phenotypes, means there is significant unmet need for new therapeutic strategies. Herein, the anti-neoplastic effects of the electrophilic fatty acid nitroalkene derivative, 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA), were investigated in multiple preclinical models of TNBC. NO2-OA reduced TNBC cell growth and viability in vitro, attenuated TNFα-induced TNBC cell migration and invasion, and inhibited the tumor growth of MDA-MB-231 TNBC cell xenografts in the mammary fat pads of female nude mice. The up-regulation of these aggressive tumor cell growth, migration, and invasion phenotypes is mediated in part by the constitutive activation of pro-inflammatory nuclear factor κB (NF-κB) signaling in TNBC. NO2-OA inhibited TNFα-induced NF-κB transcriptional activity in human TNBC cells and suppressed downstream NF-κB target gene expression, including the metastasis-related proteins intercellular adhesion molecule-1 and urokinase-type plasminogen activator. The mechanisms accounting for NF-κB signaling inhibition by NO2-OA in TNBC cells were multifaceted, as NO2-OA (a) inhibited the inhibitor of NF-κB subunit kinase β phosphorylation and downstream inhibitor of NF-κB degradation, (b) alkylated the NF-κB RelA protein to prevent DNA binding, and (c) promoted RelA polyubiquitination and proteasomal degradation. Comparisons with non-tumorigenic human breast epithelial MCF-10A and MCF7 cells revealed that NO2-OA more selectively inhibited TNBC function. This was attributed to more facile mechanisms for maintaining redox homeostasis in normal breast epithelium, including a more favorable thiol/disulfide balance, greater extents of multidrug resistance protein-1 (MRP1) expression, and greater MRP1-mediated efflux of NO2-OA–glutathione conjugates. These observations reveal that electrophilic fatty acid nitroalkenes react with more alkylation-sensitive targets in TNBC cells to inhibit growth and viability.

The ability for cells to sense and respond to microenvironmental signals is influenced by their three dimensional (3D) surroundings, which includes the extracellular matrix (ECM). In the 3D environment, vascular structures supply cells with nutrients and oxygen thus affecting cell responses such as motility. Interpretation of cell motility studies though is often restricted by the applied approaches such as 2D conventional soft lithography methods that have rectangular channel cross-sectional morphology. To better simulate cell responses to vascular supply in 3D, we developed a cell on a chip system with microfluidic channels with curved cross-sections embedded within a 3D collagen matrix that emulates anatomical vasculature more closely than inorganic polymers, thus to mimic a more physiologically relevant 3D cellular environment. To accomplish this, we constructed perfusable microfluidic channels by embedding sacrificial circular gelatin vascular templates in collagen, which were removed through temperature control. Motile breast cancer cells were pre-seeded into the collagen matrix and when presented with a controlled chemical stimulation from the artificial vasculature, they migrated towards the vasculature structure. We believe this innovative vascular 3D ECM system can be used to provide novel insights into cellular dynamics during multidirectional chemokineses and chemotaxis that exist in cancer and other diseases.

// Michelle A. Schultz 1, 2 , Andrew M. Diaz 1 , Sharon Smite 1 , Anna R. Lay 3 , Brian DeCant 1 , Ronald McKinney 1 , Windel E. Mascarinas 2 , Yinglin Xia 1 , Carola Neumann 4 , David Bentrem 2 , David W. Dawson 3 and Paul J. Grippo 1, 2 1 Division of Gastroenterology, Department of Medicine, University of Illinois-Chicago, Chicago IL 60612, USA 2 Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago IL 60611, USA 3 Department of Pathology and Laboratory Medicine and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA 90095, USA 4 Department of Pharmacology and Chemical Biology, University of Pittsburgh Medical Center, Pittsburgh PA 15232, USA Correspondence to: Michelle A. Schultz, email: buffalosoldierms@gmail.com , maschult@uic.edu Keywords: peroxiredoxin-1 (Prdx1), thioredoxin (Txn) system, Kras, pancreatic cancer Received: July 13, 2016 Accepted: June 26, 2017 Published: October 04, 2017 ABSTRACT Peroxiredoxin-1 (Prdx1), a member of the thioredoxin (Txn) system, is overexpressed and correlates with poor prognosis in pancreatic cancer patients and can suppress Kras signaling through redox-mediated inhibition of ERK and AKT in lung and breast cancer. Its redox function is maintained by Txn and sulfiredoxin (Srxn), and its tumor promoting functions are activated by post-translational modification. We studied the role of the Txn system in pancreatic neoplasia and cancer by determining how it regulates the phosphorylation of Kras effectors and by determining its association with patient survival. We found that elevated Prdx1 nuclear localization significantly correlated with better patient survival. Our data also demonstrate that the expression of the Txn system is dysregulated, with elevated Prdx1 expression and significantly decreased Txn and Srxn expression in pancreatic lesions of targeted mutant Kras mouse models. This correlated with distinct differences in the interconversion of Prdx1 oligomers that affect its ability to regulate ERK and AKT phosphorylation. Our data also suggest that Prdx1 post-translational modification and oligomerization suppress Prdx1 mediated redox regulation of ERK phosphorylation. We observed distinct differences in Txn expression and in the ability of pTyr-Prdx1 to bind to pERK in a PanIN model of pancreatic neoplasia as compared to an IPMN model, indicating a distinct difference in the function of post-translationally modified Prdx1 in cells with less Txn expression. Modified Txn system function and post-translational regulation may therefore play a significant role in pancreatic tumorigenesis by altering Kras effector phosphorylation and inhibiting the tumor suppressive redox functions of Prdx1.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Reactive oxygen and nitrogen species have cell signaling properties and are involved in a multitude of processes beyond redox homeostasis. The peroxiredoxin (Prdx) proteins are highly sensitive intracellular peroxidases that can coordinate cell signaling via direct reactive species scavenging or by acting as a redox sensor that enables control of binding partner activity. Oxidation of the peroxidatic cysteine residue of Prdx proteins are the classical post-translational modification that has been recognized to modulate downstream signaling cascades, but increasing evidence supports that dynamic changes to phosphorylation of Prdx proteins is also an important determinant in redox signaling. Phosphorylation of Prdx proteins affects three-dimensional structure and function to coordinate cell proliferation, wound healing, cell fate and lipid signaling. The advent of large proteomic datasets has shown that there are many opportunities to understand further how phosphorylation of Prdx proteins fit into intracellular signaling cascades in normal or malignant cells and that more research is necessary. This review summarizes the Prdx family of proteins and details how post-translational modification by kinases and phosphatases controls intracellular signaling.

Breastfeeding can reduce breast cancer risk; however, unknown factors modify this protective effect. Zinc (Zn) modulates an array of cellular functions including oxidative stress, cell proliferation, motility and apoptosis. Marginal Zn intake is common in women and is associated with breast cancer. We reported that marginal Zn intake in mice leads to mammary gland hypoplasia and hallmarks of pre-neoplastic lesions. In the present study, we tested the hypothesis that marginal Zn intake confounds the protective effect of lactation on breast cancer. Nulliparous mice fed control (ZA, 30 mg Zn/kg) or a marginal Zn diet (ZD, 15 mg Zn/kg), were bred and offspring were weaned naturally. Post-involution, mice were gavaged with corn oil or 7,12-dimethylbenz(a)anthracene (DMBA, 1 mg/wk for 4 weeks) and tumor development was monitored. A ZD diet led to insufficient involution, increased fibrosis and oxidative stress. Following DMBA treatment, mice fed ZD had higher oxidative stress in mammary tissue that correlated with reduced levels of peroxiredoxin-1 and p53 and tended to have shorter tumor latency and greater incidence of non-palpable tumors. In summary, marginal Zn intake creates a toxic mammary gland microenvironment and abrogates the protective effect of lactation on carcinogenesis.

Increased reactive species (RS; reactive oxygen and nitrogen species) are a byproduct of both enzymatic and non-enzymatic systems, and critical in cancer development, including breast tumorigenesis. To investigate the role of RS-related genes in breast cancer, expression levels of the most common annotated genes involved in regulating cellular RS levels and proteins that are substrates of RS in specific subtypes of breast cancer 9 were evaluated using public data bases. Based on the premise that increased RS promote tumor formation, and breast cancer subtypes vary in aggressiveness, we hypothesized that specific RS gene expression signatures are associated with breast cancer aggressiveness and patient survival. We identified a group of genes (GSTK1, PRDX2, PRDX3 and SLC36A1) that differentiate Luminal B tumors in two clusters and predict survival of patients with Luminal B breast cancers. Furthermore, network analyses of these four genes revealed an overlap of known LumB related pathways with those of RS-related signaling, which included regulation of M-phase and mitochondrial functions.

Peroxiredoxin 1 (PRDX1) senses and reduces peroxides and coordinates the signaling actions of its binding partners. We show that PRDX1 protects RAD51 from oxidation-induced repression of homologous recombination (HR) repair following ɣ-irradiation. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. The oxidation of RAD51 Cys319, a thiol located in the SH3 domain targeted by ABL, is prevented by PRDX1. Alkylation of RAD51 Cys319 by the electrophilic fatty acid nitroalkene 10- nitro-octadec-9-enoic acid (OA-NO2) inhibited RAD51-ABL protein complex formation and downstream Y315 phosphorylation. In breast cancer cells, this reactivity of OA-NO2 was manifested by diminished RAD51 foci formation and sensitization of cells to irradiation. These data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR by PRDX1 and soft electrophiles such as OA-NO2.

Peroxiredoxin 1 (PRDX1) is an oxidative stress protein sensor, which can coordinate cell signaling of its binding partners. Loss of Prdx1 function in mice has been shown to enhance susceptibility to various cancers, including breast cancer. Homologous recombination (HR) is a critical process that enables template directed DNA repair to maintain genomic stability. RAD51 is a critical component of HR, which facilitates strand-exchange to repair DNA double-strand breaks (DSB). The role of PRDX1 in DNA damage and RAD51 directed repair was investigated. Irradiation (IR) of MEFs proficient or deficient of PRDX1 with doses up to 10 Gy found Prdx1-/- MEFs were more sensitive to IR and exhibited reduced homologous recombination in DR-GFP reporter assays. Prdx1-/- MEFs also displayed enhanced DNA damage as measured by nuclear ɣH2AX phosphorylation and reduced RAD51 foci formation following IR. As PRDX1 has been previously identified to bind proteins under heightened oxidative insult, immunoprecipitation assays were conducted, which found PRDX1 directly bound RAD51 with increasing IR. Oxidation of cysteine residues to the sulfenic acid form was specifically probed with DAZ2 and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed C319 of RAD51 as a primary target of oxidation. Cells harboring overexpression of mutant RAD51 C319 revealed suppression of HR, IR-induced RAD51 foci formation, RAD51 enhanced malignancy in an anchorage-independent growth soft agar colony formation and proliferation. MDA-MB-231 breast cancer cells deficient of PRDX1 had decreased IR-induced RAD51 foci formation and were more sensitive to doxorubicin. In conclusion, PRDX1 binds RAD51 during heighted oxidative conditions and protects the functionally important RAD51 residue C319 from oxidation.

Background: The mechanisms by which stress hormones impact triple-negative breast cancer (TNBC) etiology and treatment are unclear. We have previously shown that stress hormones, cortisol, and catecholamines induce rapid DNA damage and impact DNA repair in NIH 3T3 fibroblasts. This study investigates whether stress hormones increase DNA damage in breast cancer cells and if this impacts drug efficacy. Methods: We first screened a panel of 39 breast cancer cell lines for expression of adrenergic and glucocorticoid receptors and examined if stress hormones induce DNA damage and alter cell cycle regulation in vitro. A TNBC xenograft model was used to assess the impact of restraint stress on tumour growth and chemosensitivity to paclitaxel. Results: We found that stress hormones induced DNA damage, phosphorylation of ATR, which was accompanied by an up-regulation of the G1 cell kinase inhibitor p21 and a cell cycle halt of TNBCs in the G1 phase. p21 knockdown abrogated G1 arrest by stress hormones. We also demonstrated that stress significantly decreased efficacy of paclitaxel. Conclusion: We describe a novel mechanism through which stress hormones can induce drug resistance to paclitaxel, which may have profound implications for treating drug resistance in patients with TNBC. Fil: Reeder, A.. University of Pittsburgh; Estados Unidos Fil: Attar, M.. University of Pittsburgh; Estados Unidos Fil: Nazario, L.. University of Pittsburgh; Estados Unidos Fil: Bathula, C.. University of Pittsburgh; Estados Unidos Fil: Zhang, A.. University of Pittsburgh; Estados Unidos Fil: Hochbaum, Daniel. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Roy, E.. University of Pittsburgh; Estados Unidos Fil: Cooper, K. L.. University of Pittsburgh; Estados Unidos Fil: Oesterreich, S.. University of Pittsburgh; Estados Unidos Fil: Davidson, N. E.. University of Pittsburgh; Estados Unidos Fil: Neumann, C. A.. University of Pittsburgh; Estados Unidos Fil: Flint, M. S.. University of Pittsburgh; Estados Unidos. University of Brighton; Reino Unido

Precision in redox signaling is attained through posttranslational protein modifications such as oxidation of protein thiols. The peroxidase peroxiredoxin 1 (PRDX1) regulates signal transduction through changes in thiol oxidation of its cysteines. We demonstrate here that PRDX1 is a binding partner for the tumor suppressive transcription factor FOXO3 that directly regulates the FOXO3 stress response. Heightened oxidative stress evokes formation of disulfide-bound heterotrimers linking dimeric PRDX1 to monomeric FOXO3. Absence of PRDX1 enhances FOXO3 nuclear localization and transcription that are dependent on the presence of Cys31 or Cys150 within FOXO3. Notably, FOXO3-T32 phosphorylation is constitutively enhanced in these mutants, but nuclear translocation of mutant FOXO3 is restored with PI3K inhibition. Here we show that on H2O2 exposure, transcription of tumor suppressive miRNAs let-7b and let-7c is regulated by FOXO3 or PRDX1 expression levels and that let-7c is a novel target for FOXO3. Conjointly, inhibition of let-7 microRNAs increases let-7-phenotypes in PRDX1-deficient breast cancer cells. Altogether, these data ascertain the existence of an H2O2-sensitive PRDX1-FOXO3 signaling axis that fine tunes FOXO3 activity toward the transcription of gene targets in response to oxidative stress. Antioxid. Redox Signal. 28, 62–77.

Peroxiredoxin 1 (PRDX1) senses and reduces peroxides and coordinates the signaling actions of its binding partners. Loss of Prdx1 function in mice has been shown to enhance susceptibility to various cancers, including breast cancer. Homologous recombination (HR) is a critical process that enables template directed DNA repair to maintain genomic stability. RAD51 is a critical component of HR, which facilitates strand-exchange to repair DNA double-strand breaks (DSB). The role of PRDX1 in DNA damage and RAD51 directed repair was investigated. We show that PRDX1 protects RAD51 from oxidation-induced repression of homologous recombination (HR) repair following γ-irradiation. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. PRDX1 was found to directly bind RAD51 with increasing irradiation and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed RAD51 Cys319, a thiol located in the SH3 domain targeted by ABL, as a primary target of oxidation. Pharmacological treatment with the thiol reactive electrophilic fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) targeted the C319 residue of RAD51. Alkylation of RAD51 Cys319 OA-NO2 inhibited RAD51-ABL protein complex formation and downstream Y315 phosphorylation. In breast cancer cells, this reactivity of OA-NO2 was manifested by diminished RAD51 foci formation, enhanced H2AX phosphorylation and sensitization of cells to irradiation. These data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR by PRDX1 and soft electrophiles such as OA-NO2.

Breast cancer progression and metastasis includes not only cell-autonomous properties of cancer epithelial cells, but also the influence of the neighboring tumor stromal cells. In breast cancer, almost 80% of stromal associated fibroblasts (SAFs) acquire a cancer associated fibroblast (CAF)-like “activated phenotype”. This CAF activated phenotype is associated with elevated levels of reactive oxygen species (ROS) that are linked with tumor remodeling and spreading. Members of the lysyl oxidase (LOX) family of enzymes participate in tumor remodeling through the promotion of collagen crosslinking and collagen fibril production. We hypothesize that stromal Prdx1 regulates ROS dependent metastasis/migration of cancer cells through effects on LOX activity. Our preliminary data reveal that Prdx1 prevents CAF-induced malignant phenotypes in breast cancer (epithelial) cells in an H2O2-dependent manner. When compared to wild-type mice, Prdx1-/- SAFs show a marked increase in CAF-specific characteristics, including increased expression of CAF-specific markers, motility and invasiveness of SAFs and SAF-induced chemotactic migration and invasion by breast cancer epithelial cells in vitro. Lack of Prdx1 in mammary SAFs results in the upregulation of markers of the activated phenotype, such as collagen, vimentin and α-SMA leading to an increase in co-migration and invasion. As shPrdx1 SAFs show CAF-like mesenchymal properties in vitro, we tested in vivo if Prdx1 suppresses migration of breast cancer cells by generating a syngeneic mouse model to image BALB/c derived SAFs shPrdx1/SAFs pLKO1 (expressing iRFP).

Recent studies suggest that Peroxiredoxin 1 (Prdx1), in addition to its known H₂O₂-scavenging function, mediates cell signaling through redox-specific protein-protein interactions. Our data illustrate how Prdx1 specifically coordinates p38MAPK-induced signaling through regulating p38MAPKα phosphatases in an H₂O₂ dose-dependent manner. MAPK phosphatases (MKP-1 and/or MKP-5), which are known to dephosphorylate and deactivate the senescence-inducing MAPK p38α, belong to a group of redox-sensitive phosphatases (protein tyrosine phosphatases) characterized by a low pKa cysteine in their active sites. We found that Prdx1 bound to both MKP-1 and MKP-5, but dissociated from MKP-1 when the Prdx1 peroxidatic cysteine Cys52 was over-oxidized to sulfonic acid, which in turn resulted in MKP-1 oxidation-induced oligomerization and inactivity toward p38MAPKα. Conversely, over-oxidation of Prdx1-Cys52 was enhancing in the Prdx1:MKP-5 complex with increasing amounts of H₂O₂ concentrations and correlated with a protection from oxidation-induced oligomerization and inactivation of MKP-5 so that activation toward p38MAPK was maintained. Further examination of this Prdx1-specific mechanism in a model of reactive oxygen species-induced senescence of human breast epithelial cells revealed the specific activation of MKP-5, resulting in decreased p38MAPKα activity. Taken together, our data suggest that Prdx1 orchestrates redox signaling in an H₂O₂ dose-dependent manner through the oxidation status of its peroxidatic cysteine Cys52.

Prostate cancer (PCa) is the most frequently diagnosed malignancy in men, and the second highest contributor of male cancer related lethality. Disease mortality is due primarily to metastatic spread, highlighting the urgent need to identify factors involved in this progression. Activation of the genetic epithelial to mesenchymal transition (EMT) program is implicated as a major contributor of PCa progression. Initiation of EMT confers invasive and metastatic behavior in preclinical models and is correlated with poor clinical prognosis. Extracellular Hsp90 (eHsp90) promotes cell motility and invasion in cancer cells and metastasis in preclinical models, however, the mechanistic basis for its widespread tumorigenic function remains unclear. We have identified a novel and pivotal role for eHsp90 in driving EMT events in PCa. In support of this notion, more metastatic PCa lines exhibited increased eHsp90 expression relative to their lineage-related nonmetastatic counterparts. We demonstrate that eHsp90 promoted cell motility in an ERK and matrix metalloproteinase-2/9-dependent manner, and shifted cellular morphology toward a mesenchymal phenotype. Conversely, inhibition of eHsp90 attenuated pro-motility signaling, blocked PCa migration, and shifted cell morphology toward an epithelial phenotype. Last, we report that surface eHsp90 was found in primary PCa tumor specimens, and elevated eHsp90 expression was associated with increased levels of matrix metalloproteinase-2/9 transcripts. We conclude that eHsp90 serves as a driver of EMT events, providing a mechanistic basis for its ability to promote cancer progression and metastasis in preclinical models. Furthermore, its newly identified expression in PCa specimens, and potential regulation of pro-metastatic genes, supports a putative clinical role for eHsp90 in PCa progression.

The loss of the H(2)O(2) scavenger protein encoded by Prdx1 in mice leads to an elevation of reactive oxygen species (ROS) and tumorigenesis of different tissues. Loss of heterozygosity (LOH) mutations could initiate tumorigenesis through loss of tumor suppressor gene function in heterozygous somatic cells. A connection between the severity of ROS and the frequency of LOH mutations in vivo has not been established. Therefore, in this study, we characterized in vivo LOH in ear fibroblasts and splenic T cells of 3-4 month old Prdx1 deficient mice. We found that the loss of Prdx1 significantly elevates ROS amounts in T cells and fibroblasts. The basal amounts of ROS were higher in fibroblasts than in T cells, probably due to a less robust Prdx1 peroxidase activity in the former. Using Aprt as a LOH reporter, we observed an elevation in LOH mutation frequency in fibroblasts, but not in T cells, of Prdx1(-/-) mice compared to Prdx1(+/+) mice. The majority of the LOH mutations in both cell types were derived from mitotic recombination (MR) events. Interestingly, Mlh1, which is known to suppress MR between divergent sequences, was found to be significantly down-regulated in fibroblasts of Prdx1(-/-) mice. Therefore, the combination of elevated ROS amounts and down-regulation of Mlh1 may have contributed to the elevation of MR in fibroblasts of Prdx1(-/-) mice. We conclude that each tissue may have a distinct mechanism through which Prdx1 deficiency promotes tumorigenesis.

Purpose MicroRNAs (miRNAs) have emerged as critical regulators of many cellular pathways. Ionizing radiation (IR) exposure causes DNA damage and induces premature senescence. However, the role of miRNAs in IR-induced senescence has not been well defined. Thus, the purpose of this study was to identify and characterize senescence-associated miRNAs (SA-miRNAs) and to investigate the role of SA-miRNAs in IR-induced senescence. Methods and Materials In human lung (WI-38) fibroblasts, premature senescence was induced either by IR or busulfan (BU) treatment, and replicative senescence was accomplished by serial passaging. MiRNA microarray were used to identify SA-miRNAs, and real-time reverse transcription (RT)-PCR validated the expression profiles of SA-miRNAs in various senescent cells. The role of SA-miRNAs in IR-induced senescence was characterized by knockdown of miRNA expression, using anti-miRNA oligonucleotides or by miRNA overexpression through the transfection of pre-miRNA mimics. Results We identified eight SA-miRNAs, four of which were up-regulated (miR-152, -410, -431, and -493) and four which were down-regulated (miR-155, -20a, -25, and -15a), that are differentially expressed in both prematurely senescent (induced by IR or BU) and replicatively senescent WI-38 cells. Validation of the expression of these SA-miRNAs indicated that down-regulation of miR-155, -20a, -25, and -15a is a characteristic miRNA expression signature of cellular senescence. Functional analyses revealed that knockdown of miR-155 or miR-20a, but not miR-25 or miR-15a, markedly enhanced IR-induced senescence, whereas ectopic overexpression of miR-155 or miR-20a significantly inhibited senescence induction. Furthermore, our studies indicate that miR-155 modulates IR-induced senescence by acting downstream of the p53 and p38 mitogen-activated protein kinase (MAPK) pathways and in part via regulating tumor protein 53-induced nuclear protein 1 (TP53INP1) expression. Conclusion Our results suggest that SA-miRNAs are involved in the regulation of IR-induced senescence, so targeting these miRNAs may be a novel approach for modulating cellular response to radiation exposure.

Autophagy is activated in response to cellular stressors and mediates lysosomal degradation and recycling of cytoplasmic material and organelles as a temporary cell survival mechanism. Defective autophagy is implicated in human pathology, as disruption of protein and organelle homeostasis enables disease-promoting mechanisms such as toxic protein aggregation, oxidative stress, genomic damage, and inflammation. We previously showed that autophagy-defective immortalized mouse mammary epithelial cells are susceptible to metabolic stress, DNA damage, and genomic instability. We now report that autophagy deficiency is associated with endoplasmic reticulum (ER) and oxidative stress, and with deregulation of p62-mediated keratin homeostasis in mammary cells, allograft tumors, and mammary tissues from genetically engineered mice. In human breast tumors, high phospho(Ser73)-K8 levels are inversely correlated with Beclin 1 expression. Thus, autophagy preserves cellular fitness by limiting ER and oxidative stress, a function potentially important in autophagy-mediated suppression of mammary tumorigenesis. Furthermore, autophagy regulates keratin homeostasis in the mammary gland via a p62-dependent mechanism. High phospho(Ser73)-K8 expression may be a marker of autophagy functional status in breast tumors and, as such, could have therapeutic implications for breast cancer patients. Mol Cancer Res; 8(6); 873–84. ©2010 AACR.

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS-induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras-induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation-induced inactivation. Along those lines, Prdx1 tumour suppression of Ras- or ErbB-2-induced transformation was mediated mainly via PTEN.

It has been known for many years that free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), promote diseases such as cancer. Peroxiredoxins (Prdxs) are small H(2)O(2) scavenging proteins that appear to have tumor preventive functions since loss of Prdx1 in mice leads to premature death from cancer. However, as Prdxs are antioxidants they also scavenge the H(2)O(2) in cancer cells that way supporting survival and tumor maintenance. This suggests that Prdxs function as tumor 'preventers' rather than as tumor suppressors since they do not induce cell death when re-expressed in cancer cells, as it occurs with the tumor suppressor p53. Therefore, the knowledge of Prdx function and regulation may help provide a fuller understanding of the role of ROS in tumorigenesis.

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising

The progression of mammalian cells through the G1 phase of the cell cycle is driven by the D-type and E-type cyclins (43). These cyclins bind, activate, and provide substrate specificity for their associated cyclin-dependent kinases (CDKs). In contrast to other cyclins, which are induced periodically during cell cycle progression, the expression of D cyclins is controlled largely by the extracellular environment. For this reason, D cyclins are regarded as links between the external mitogenic milieu and the core cell cycle machinery (39, 45). Three D-type cyclins, D1, D2 and D3, have been enumerated in mammalian cells (21, 33, 34, 37, 38, 57). These three proteins are encoded by separate genes located on different chromosomes, but they show significant amino acid similarity, suggesting that they arose from a common primordial ancestor gene (19, 58). On average, D cyclins show 50 to 60% identity throughout the entire coding sequence and 75 to 78% identity within the most conserved cyclin box domain (19, 58). All three D cyclins associate with CDK4 or CDK6, yielding six different combinations of cyclin D-CDK holoenzymes (2, 10, 20, 31, 32, 36). An important issue is whether each of the D cyclins performs unique, possibly cell type-specific functions or the three proteins represent tissue-specific isoforms with virtually identical functions. At a biochemical level, all three D cyclins were shown to physically associate with CDK4 and CDK6 and to drive phosphorylation of the retinoblastoma protein, pRB, and pRB-related “pocket” proteins p107 and p130 (3, 28, 32, 36, 54, 56). The phosphorylation of these pocket proteins may represent the major function for cyclin D-CDK complexes in cell cycle progression, as shown by the observations that cells lacking pRB or p107 and p130 no longer require D cyclins for proliferation (1, 4, 16, 23, 29, 35, 40, 53). However, biochemical differences between the three D cyclins were noted. Thus, cyclins D2 and D3 can form active complexes with CDK2, while cyclin D1 was reported to lack this ability (10, 17). Moreover, in addition to their well-established CDK-dependent functions, D cyclins were shown to interact with tissue-specific transcription factors, such as estrogen receptor, androgen receptor, thyroid receptor, and retinoic acid receptor alpha, C/EBP binding protein β, DMP1, and others (8, 27). In some cases, this interaction was uniquely ascribed to a particular D-type cyclin (9, 60). To address the functions of the D-type cyclins in development, we and others generated mice lacking cyclin D1, D2, or D3 and characterized their phenotypes (12, 46-48). We found that mice lacking individual D cyclins were viable and displayed narrow, tissue-specific abnormalities. For instance, cyclin D1-deficient mice showed underdeveloped, hypoplastic retinas and presented a developmental neurological abnormality. Moreover, cyclin D1-deficient females displayed a normal mammary epithelial tree at the end of sexual maturation, but they failed to undergo full lobuloalveolar development during pregnancy (12, 48). Importantly, all these compartments developed normally in cyclin D2- or D3-deficient animals (46, 47), revealing a unique requirement for cyclin D1 in vivo in selected tissues. In the present study, we asked whether the requirement for cyclin D1 function in these compartments was caused by tissue-specific pattern of D cyclin expression or alternatively reflected the presence of specialized tissue-specific functions for cyclin D1. To address this question by genetic means, we generated a knock-in strain of mice expressing cyclin D2 in place of cyclin D1. We next asked whether cyclin D2 could drive the normal development of cyclin D1-dependent tissues.

Breast cancer is a highly complex tissue composed of neoplastic and stromal cells. Carcinoma-associated fibroblasts (CAFs) are commonly found in the cancer stroma, where they promote tumor growth and enhance vascularity in the microenvironment. Upon exposure to oxidative stress, fibroblasts undergo activation to become myofibroblasts. These cells are highly mobile and contractile and often express numerous mesenchymal markers. CAF activation is irreversible, making them incapable of being removed by nemosis. In breast cancer, almost 80% of stromal fibroblasts acquire an activated phenotype that manifests by secretion of elevated levels of growth factors, cytokines, and metalloproteinases. They also produce hydrogen peroxide, which induces the generation of subsequent sets of activated fibroblasts and tumorigenic alterations in epithelial cells. While under oxidative stress, the tumor stroma releases high energy nutrients that fuel cancer cells and facilitate their growth and survival. This review describes how breast cancer progression is dependent upon oxidative stress activated stroma and proposes potential new therapeutic avenues.

Sphingosine kinase 1 (SK1) is an important enzyme involved in the production of the bioactive lipid sphingosine 1-phosphate (S1P). SK1 is overexpressed in many forms of cancer, however, the contribution of SK1 to cancer progression is still unclear. One of the best characterized mutations found in several forms of human cancer is an activating point mutation in the Ras oncogene, which disrupts its GTPase activity and leads to stimulation of the MEK/ERK pathway. Because SK1 activity and subcellular localization have been shown to be regulated by ERK, we wished to investigate the effect of oncogenic Ras, a potent activator of the Raf/MEK/ERK pathway, on the activity of SK1 and sphingolipid metabolism. Using HEK293T cells transiently transfected with the K-RasG12V oncogene and both wild type and Sphk1(-/-) mouse embryonic fibroblasts stably infected with retroviral K-RasG12V, we found that K-RasG12V increases the production of S1P and decreases the production of ceramide in a SK1-dependent manner. In addition, we found that expression of the K-RasG12V oncogene leads to plasma membrane localization of SK1 and a reduction in cytosolic levels of SK1. This effect is likely mediated by the Raf/MEK/ERK pathway as constitutively active B-Raf or MEK1 are able to activate SK1, but constitutively active Akt1 is not. We believe this research has important implications for how sphingolipids may be contributing to oncogenic transformation and provide some of the first evidence for oncogenes inducing specific changes in sphingolipid metabolism through SK1 regulation.

More than half of pygmy sperm whales (Kogia breviceps) that strand exhibit signs of cardiomyopathy (CMP). Many factors may contribute to the development of idiopathic CMP in K. breviceps, including genetics, infectious agents, contaminants, biotoxins, and dietary intake (e.g. selenium, mercury, and pro-oxidants). This study assessed trace elements in K. breviceps at various stages of CMP progression using fresh frozen liver and heart samples collected from individuals that stranded along US Atlantic and Gulf coasts between 1993 and 2007. Standard addition calibration and collision cell inductively coupled plasma mass spectrometry (ICP-MS) were employed for total Se analysis and pyrolysis atomic absorption (AA) was utilized for total Hg analysis to examine if the Se/Hg detoxification pathway inhibits the bioavailability of Se. Double spike speciated isotope dilution gas chromatography ICP-MS was utilized to measure methyl Hg and inorganic Hg. Immunoblot detection and colorimetric assays were used to assess protein oxidation status. Data collected on trace elements, selenoproteins, and oxidative status were evaluated in the context of animal life history and other complementary histological information to gain insight into the biochemical pathways contributing to the development of CMP in K. breviceps. Cardiomyopathy was only observed in adult pygmy sperm whales, predominantly in male animals. Both Hg:Se molar ratios and overall protein oxidation were greater in males than females and increased with progression of CMP.

Peroxiredoxins (Prdxs) are a family of small (22–27 kDa) nonseleno peroxidases currently known to possess six mammalian isoforms. Although their individual roles in cellular redox regulation and antioxidant protection are quite distinct, they all catalyze peroxide reduction of H(2)O(2), organic hydroperoxides and peroxynitrite.(1,2) They are found to be expressed ubiquitously and in high levels,(3) suggesting that they are both an ancient and important enzyme family. Prdxs can be divided into three major subclasses: typical 2-cysteine (2-Cys) Prdxs (Prdx1–4), atypical 2-Cys Prdx (Prdx 5) and 1-Cys Prdx (Prdx 6). Recent evidence suggests that 2-Cys peroxiredoxins are more than “just simple peroxidases”. This hypothesis has been discussed elegantly in recent review articles, considering “over”-oxidation of the protonated thiolate peroxidatic cysteine and post-translational modification of Prdxs as processes initiating a mechanistic switch from peroxidase to chaperon function.(4–6) The process of over-oxidation of the peroxidatic cysteine (C(P)) occurs during catalysis in the presence of thioredoxin (Trx), thus rendering the sulfenic moiety to sulfinic acid,(7) which can be reduced by sulfiredoxin (Srx).(8,9) However, further oxidation to sulfonic acid is believed to promote Prdx degradation or, as recently shown, the formation of oligomeric peroxidase-inactive chaperones(10) with questionable H(2)O(2)-scavenging capacity. In the light of this and given that Prdx1 has recently been shown by us(11) and by others(12–17) to interact directly with signaling molecules, we will explore the possibility that H(2)O(2) regulates signaling in the cell in a temporal and spatial fashion via oxidizing Prdx1. Therefore, this review will focus on H(2)O(2) modulating cell signaling via Prdxs by discussing: (1) the activity of Prdxs towards H(2)O(2); (2) sub cellular localization and availability of other peroxidases, such as catalase or glutathione peroxidases; (3) the availability of Prdxs reducing systems, such as thioredoxin and sulfiredoxin and lastly, (4) Prdx1 interacting signaling molecules.

The peroxiredoxin (Prdx) family of antioxidant enzymes uses redox-active cysteines to reduce peroxides, lipid hydroperoxides, and peroxynitrites. Prdx1 is known to be important to protect red blood cells against reactive oxygen species and in tumor prevention. In this study, the role of Prdx1 in inflammation, thrombosis, and atherosclerosis was investigated. Using intravital microscopy, we showed that the number of leukocytes rolling per minute in unstimulated veins was increased by 2.5-fold in Prdx 1 −/− compared to Prdx 1 +/+ mice. In Prdx 1 −/− mice, 50% of leukocytes rolled at a velocity Prdx 1 +/+ mice, suggesting that adhesion molecule density on the endothelium may have been increased by Prdx1 deficiency. Indeed, endothelial P-selectin, soluble P-selectin, and von Willebrand factor in plasma were increased in Prdx 1 −/− mice compared to Prdx 1 +/+ mice, indicating elevated Weibel–Palade body release. In contrast to this excessive endothelial activation, Prdx 1 −/− platelets showed no sign of hyperreactivity, and their aggregation both in vitro and in vivo was normal. We also examined the role of Prdx1 in the apoE −/− murine spontaneous model of atherosclerosis. Prdx 1 −/− / apoE −/− mice fed normal chow developed larger, more macrophage-rich aortic sinus lesions than Prdx 1 +/+ / apoE −/− mice, despite similar amounts and size distributions of cholesterol in their plasma lipoproteins. Thus, Prdx1 protects against excessive endothelial activation and atherosclerosis, and the Prdx 1 −/− mice could serve as an animal model susceptible to chronic inflammation.

8p11 myeloproliferative syndrome (EMS) is a hematopoietic stem cell disorder characterized by myeloid hyperplasia and non-Hodgkin's lymphoma with chromosomal translocations fusing several genes, most commonly ZNF198, to fibroblast growth factor receptor-1 (FGFR1). However, patients with BCR-FGFR1 fusion present with typical chronic myeloid leukemia (CML). We demonstrate that ZNF198-FGFR1 induces EMS-like disease in mice, with myeloproliferation and T lymphoma arising from common multipotential progenitors. Mutation of FGFR1 Tyr766 attenuates both myeloid and lymphoid diseases, identifying phospholipase C-γ1 as a downstream effector. Bcr-FGFR1 binds Grb2 via Bcr Tyr177 and induces CML-like leukemia in mice, whereas Bcr-FGFR1/Y177F lacks Grb2 binding and causes EMS-like disease. These results implicate different signaling pathways originating from both kinase and fusion partner in the pathogenesis of CML and EMS.

Prostate cancer (PCa) is the most frequently diagnosed malignancy and second most common cause of cancer related lethality in men. Disease mortality is due primarily to metastatic spread, highlighting the urgent need to identify factors involved in this progression. Increased secretion of heat shock protein 90 (Hsp90) has been reported in diverse cancer cells, and secreted, or extracellular Hsp90 (eHsp90) is also found in patient plasma. Although eHsp90 promotes cancer cell motility and invasion and facilitates tumor metastasis, the mechanistic basis for its activity has remained unknown. The goal of this study was to explore whether extracellular Hsp90 (eHsp90) may play a role in PCa progression. To explore this, 3 sets of lineage-related differentially metastatic cell pairs were utilized to evaluate eHsp90 expression and activity. We found that eHsp90 secretion correlated with PCa aggressiveness. To evaluate the functional role of eHsp90, we modulated its expression and activity. Upregulation of eHsp90 was accomplished by exposure of prostate epithelial cells to exogenous Hsp90 protein, or alternatively, cells were transduced with a lentivirus directing the secretion of Hsp90. Blockade of eHsp90 function was accomplished by exposure of cells to anti-Hsp90 antibodies or to a cell-impermeant specific inhibitor of Hsp90. We demonstrate that eHsp90 is a critical factor required for PCa cell motility and for initiation of the epithelial to mesenchymal transition (EMT) genetic program. Moreover, modest increases in eHsp90 were sufficient to induce tumor growth and localized invasion in a subrenal capsule xenograft model of PCa. Finally, eHsp90 was detected in human cancer specimens and its expression was correlated with upregulated expression of pro-invasive genes. Our findings offer insights into the mechanistic basis of eHsp90 in PCa, and further support a putative clinical role for eHsp90 as a potential driver of PCa progression. Citation Format: Michael W. Hance, Krystal Nolan, Udhayakumar Gopal, Jessica E. Bohonowych, Agnieszka Jezierska-Drutel, Carola A. Neumann, Haibo Liu, Garraway P. Isla, Omar E. Franco, Simon W. Hayward, Jennifer S. Isaacs. Tumor secreted Hsp90 initiates tumor growth and the epithelial to mesenchymal transition (EMT) in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A74.

Crosstalk between mammary epithelium and stromal cell types, including fibroblasts and adipocytes is 1) essential for mammary gland development 2) likely impacts breast cancer potential and 3) significantly impacts the growth, invasion and metastasis of tumor cells. Although well recognized, molecular mechanisms important in stromal-epithelial crosstalk are still poorly understood. The purpose of this study is to better elucidate one potentially significant mechanism involving stromal fibroblast to epithelial paracrine signaling in the mammary gland. We previously demonstrated that HoxC6 was required for mammary epithelial cell growth and ductal branching morphogenesis using mouse models and 3-dimensional in vitro culture of human mammary epithelial cell lines. We also demonstrated that constitutive ErbB2/Her2/Neu receptor activation in epithelium of transgenics is sufficient to rescue HoxC6 deficiency in epithelial growth but not branching. Significantly, this model also shows that HoxC6 null stroma facilitates tumor metastasis. Here, we focus attention on stromal fibroblasts (vimentin positive cells) from wild type and HoxC6 null prepubertal mouse thoracic mammary glands (TMGs). When cultured in vitro, HoxC6 null TMG fibroblasts display multiple filopodia and extended cellular processes that could indicate an activated myofibroblast phenotype. In contrast, cultured wild type TMG fibroblasts appear quite similar to mouse embryonic fibroblasts. Quantitative comparisons of gene expression patterns in isolated fibroblasts indicate that HoxC6 null fibroblasts are more activated than wild type fibroblasts. Activation markers studied include alpha smooth muscle actin (ACTA2/SMA), S100A4 (Fsp1) and Wnt1-induced secreted protein 1 (Wisp1). Since estrogens are know to repress HoxC6 expression and are implicated in modulating paracrine stromal-to-epithelial interactions, stromal fibroblasts were isolated and cultured under estrogen-free conditions and treated with 17beta-estradiol, bisphenol A and vitamin D. In addition, stromal fibroblasts were co-cultured with mammary epithelial stem cells and stem-like mammary tumor cells to study the impact of stromal fibroblast HoxC6 status on epithelial cell activities. The results obtained will be discussed as they pertain to stromal-to-epithelial signaling in mammary gland development and in the growth, invasion and metastasis of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5267.

Abstract #2010 Background: Early menarche and breast development are increasingly common risk factors for breast cancer. Epigenetic mechanisms implicated include natural and anthropogenic steroids, such as estrogens, that disrupt endocrine signaling and dysregulate stem cell homeostasis. Animal models show that, although a proliferative response to estrogens in mammary epithelium is not observed until near puberty, in utero or neonatal exposure can recapitulate aberrant breast development. The molecular basis for the delay in proliferative response remains poorly understood and assessments of agent activity, potency and risk remain inaccurate. Here, we provide a candidate molecular mechanism involving the homeobox transcription factor HoxC6, through which more accurate assessments may be facilitated. Materials & Methods: Genetically and hormonally manipulated in vivo mouse models (HoxC6 targeted disruption, C6KO; and mammary epithelial-specific inducible HoxC6, MMTV-rtTA TRE-HoxC6-IRES-EGFP) and in vitro human breast cancer cell lines (MCF10A, MCF7 and MDA-MB-231; "231") were used to assess impacts on mammary epithelial growth, differentiation and expression of HoxC6 and its direct target genes IGFBP3, CD44 and FGFR2. Cell lines were manipulated using HoxC6 shRNA and cDNA lentivirus and activated Akt isoform retroviral expression vectors. Gene expression and signaling were measured by quantitative RT-qPCR, IHC, Western blot and using the BioPlex200 Multiplex System. Results: RT-qPCR analysis shows HoxC6 expression elevated in well-differentiated cell lines (MCF10A, 5.06X; MCF7, 5.16X) and decreased in more aggressive tumor cell lines (-1.95X, 231 cells; relative to A549 cells). HoxC6 knockdown in MCF10A cells results in an inhibition of cellular growth and decreased branching in 3D cultures and dysregulation of HoxC6 target genes. These results are consistent with in vivo results in which C6KO causes defective postnatal mammary epithelial growth/branching. Rescue of growth defects in knockdown MCF10A cells is achieved to varying extents with myristoylated Akt isoforms, however morphological defects persist and are associated with EMT. HoxC6 induction in stem cells is associated with altered self-renewal and differentiation. HoxC6 repression by estrogens was assessed in vivo under conditions of neonatal estrogen exposure and estrogen-free diets, showing alterations in HoxC6 and target gene expressions and other phenotypic changes. Discussion: Based on our results, we will present a model in which HoxC6 is estrogen-responsive and coordinates mammary stem/progenitor cell homeostatic pathways, including FGF and GH-IGF1 signaling in the control of pool size and branching morphogenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2010.