Kathryn N. Phoenix

Double-Stranded RNA-Binding Protein Regulates Vascular Endothelial Growth Factor mRNA Stability, Translation, and Breast Cancer Angiogenesis

ABSTRACT Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3′ untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo.

Dietary energy availability affects primary and metastatic breast cancer and metformin efficacy

Dietary energy restriction has been shown to repress both mammary tumorigenesis and aggressive mammary tumor growth in animal studies. Metformin, a caloric restriction mimetic, has a long history of safe use as an insulin sensitizer in diabetics and has been shown to reduce cancer incidence and cancer-related mortality in humans. To determine the potential impact of dietary energy availability and metformin therapy on aggressive breast tumor growth and metastasis, an orthotopic syngeneic model using triple negative 66cl4 tumor cells in Balb/c mice was employed. The effect of dietary restriction, a standard maintenance diet or a diet with high levels of free sugar, were tested for their effects on tumor growth and secondary metastases to the lung. Metformin therapy with the various diets indicated that metformin can be highly effective at suppressing systemic metabolic biomarkers such as IGF-1, insulin and glucose, especially in the high energy diet treated animals. Long-term metformin treatment demonstrated moderate yet significant effects on primary tumor growth, most significantly in conjunction with the high energy diet. When compared to the control diet, the high energy diet promoted tumor growth, expression of the inflammatory adipokines leptin and resistin, induced lung priming by bone marrow-derived myeloid cells and promoted metastatic potential. Metformin had no effect on adipokine expression or the development of lung metastases with the standard or the high energy diet. These data indicate that metformin may have tumor suppressing activity where a metabolic phenotype of high fuel intake, metabolic syndrome, and diabetes exist, but may have little or no effect on events controlling the metastatic niche driven by proinflammatory events.

AMP-Activated Protein Kinase α 2 Isoform Suppression in Primary Breast Cancer Alters AMPK Growth Control and Apoptotic Signaling

Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic regulator that promotes energy conservation and restoration when cells are exposed to nutrient stress. Given the high metabolic requirement of cancer cells, AMPK activation has been suggested as a potential preventative and therapeutic target. However, previous findings have shown that AMPK activity is diminished in some cancers. Expression of the 2 catalytic isoforms, AMPKα1 and AMPKα2, was evaluated in primary breast cancer and matched nontumor-adjacent tissue samples using immunohistochemistry. AMPK-dependent growth signaling events were examined in primary human mammary epithelial cells (HMECs) using RNAi to understand the importance of AMPKα2 in normal growth regulation. To test whether AMPKα2 would reinstate growth control and apoptotic mechanisms in breast cancer cells, metabolic stress assays and tumor xenografts were performed in MCF-7 cells, expressing low levels of AMPKα2, with stable transfection of either green fluorescent protein (GFP) or AMPKα2 expression constructs. AMPKα2 was found to be significantly suppressed in breast cancer tissue samples, whereas AMPKα1 was not. In normal HMECs, low glucose stress resulted in AMPK-driven growth inhibition. Interestingly, this response was ablated when AMPKα2 was silenced. Metabolic stress assays in MCF-7 cells indicated that AMPKα2 expression reduced both mTOR signaling and cyclin D1 expression, contributing to G1-phase cell cycle arrest. Cells expressing AMPKα2 underwent apoptosis more readily than GFP control cells. Xenograft studies demonstrated that MCF-7 tumors expressing AMPKα2 display reduced proliferation and increased apoptotic events. Furthermore, AMPKα2 xenografts exhibited diminished cyclin D1 levels along with an increased amount of nuclear p53, thereby implicating the AMPKα2-p53 signaling axis as a mediator of cell apoptosis. Together, these results highlight the significance of reduced AMPK activity contributing to human carcinogenesis and, specifically, the role of AMPKα2 with respect to its control of normal mammary epithelial cell growth and its reduced expression in breast cancer.

AMPKα2 Suppresses Murine Embryonic Fibroblast Transformation and Tumorigenesis

AMP-activated kinase (AMPK) is a key metabolic sensor and stress signaling kinase. AMPK activity is known to suppress anabolic processes such as protein and lipid biosynthesis and promote energy-producing pathways including fatty acid oxidation, resulting in increased cellular energy. In addition, AMPK localizes to centrosomes during cell division, plays a role in cellular polarization, and directly targets p53, affecting apoptosis. Two distinct catalytic AMPKα isoforms exist: α1 and α2. Multiple reports indicate that both common and distinct functions exist for each of the 2 α isoforms. AMPK activation has been shown to repress tumor growth, and it has been suggested that AMPK may function as a metabolic tumor suppressor. To evaluate the potential role of each of the AMPKα isoforms in modulating cellular transformation, susceptibility to Ras-induced transformation was evaluated in normal murine embryonic fibroblasts (MEFs) obtained from genetically deleted AMPKα1- or AMPKα2-null mice. This study demonstrated that while AMPKα1 is the dominant AMPK isoform expressed in MEFs, only the AMPKα2-null MEFs displayed increased susceptibility to H-RasV12 transformation in vitro and tumorigenesis in vivo. Conversely, AMPKα1-null MEFs, which demonstrated compensation with increased expression of AMPKα2, displayed minimal transformation susceptibility, decreased cell survival, decreased cell proliferation, and increased apoptosis. Finally, this study demonstrates that AMPKα2 was selectively responsible for targeting p53, thus contributing to the suppression of transformation and tumorigenic mechanisms.

Revealing the role of phospholipase Cβ3 in the regulation of VEGF-induced vascular permeability.

VEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cβ3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCβ3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.

Identification of Novel Tumor Antigens With Patient-Derived Immune-Selected Antibodies

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions.

Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors

BackgroundMonoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this approach led to identification of a tumor specific single domain antibody. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells.MethodsEight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2.ResultsFlow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and preferential binding to human breast tumor tissues in comparison to normal breast tissues. In primary breast tumor tissues, HCAb2 showed positive binding to both E-cadherin positive and negative tumor cells. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells.ConclusionsFrom our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody.

Abstract P4-08-01: AMPK facilitates breast cancer cell survival by modulating microenvironmental stress

Recurrent and metastatic breast cancers are responsible for the majority of breast-cancer related deaths. These cancer cells are able to adapt to stressors within the tumor microenvironment including hypoxia, low nutrient levels, and chemotherapy-induced toxicities. Breast cancer cells can respond to these microenvironmental stressors through a variety of mechanisms, including cell cycle inhibition and metabolic alteration. Tumor cell survival is dependent on the ability to alter these mechanisms in response to stress. AMPK (AMP-activated protein kinase) is the main metabolic sensor of the cell, and both its expression and activity have been reported to be altered in breast cancer. Moreover, there are two isoforms of the catalytic subunit (α1 and α2), and differential functionality of these isoforms has been suggested. Using estrogen receptor-positive human breast cancer cell lines, we investigated the effect of differential AMPKα isoform expression on breast cancer cell survival. We found that over-expression of AMPKα2 in MCF-7 cells resulted in decreased ATP production in response to low glucose levels, while the knockdown of AMPKα2 in HCC1500 cells ablated this response to low glucose conditions. A similar difference in response was also seen when the cells were treated with a combination of nutrient stress and the estrogen receptor alpha (ERα) inhibitor, ICI182780. In response to this finding, we compared the glycolytic and oxygen consumption rates of our MCF-7 GFP and MCF-7 AMPKα2 cells. We found that in response to low glucose stress, AMPKα2 expressing MCF-7 cells maintained both a higher glycolytic rate and a higher oxygen consumption rate as compared to GFP cells. Furthermore, these cells seem to alter their cellular signaling in response to metabolic stress faster than GFP cells. To evaluate this differential response to microenvironmental stress in vivo, MCF-7 cells expressing either GFP or AMPKα2 were injected into athymic nude mice previously implanted with slow-release estradiol pellets. After one week, the estradiol pellets were removed to induce cellular dormancy for thirty days. Analysis of tumors at this time indicated that more of the AMPKα2 expressing cells survived estradiol deprivation than did the control cells. Analysis of proliferation by Ki67 staining indicated that the GFP cells maintained proliferation during deprivation, while AMPKα2 cells were largely negative for proliferation. ApoTag staining revealed a similar trend for apoptotic cells. This suggests that an inability to control cell cycle resulted in a decreased survival of the GFP cells under estradiol deprivation. Following the deprivation period, estradiol pellets were re-implanted and residual dormant tumors resumed growth. AMPKα2 tumors grew to roughly double the size of GFP tumors. Interestingly, AMPKα2 tumors had a higher number of mitotic events than did GFP tumors as visualized by Ki67 staining. This could be due to more viable cells being present following estradiol deprivation. We conclude that the expression of AMPKα2 promotes long-term breast cancer survival in estrogen-sensitive cells, due to their increased ability to sense and respond to changes in their microenvironment, which therefore increases their chances for survival. Citation Format: Sullivan KL, Kopsiaftis S, Phoenix KN, Fox MM, Tsurutani N, Vella AT, Claffey KP. AMPK facilitates breast cancer cell survival by modulating microenvironmental stress. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-01.

Abstract 1138: AMPK promotes survival of breast cancer cells by modulating metabolic stress

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Breast cancer cells can respond to microenvironmental stressors by becoming dormant, that is inhibiting cell proliferation until the environment becomes growth-permissive. One of the survival pathways implicated in dormant cancer cells is the p38MAPK pathway. Modulation of signaling to combat metabolic stressors could provide survival benefits to dormant breast cancer cells. AMPK (AMP-activated protein kinase) is the central metabolic regulator of the cell, and its expression is altered in breast cancer. Moreover, there are two isoforms of the catalytic subunit (α1 and α2), and differential functionality of these isoforms has been reported. Using estrogen-receptor positive human breast cancer cell lines, we investigated the effect of differential AMPKα isoform expression on breast cancer survival. We found that over-expression of AMPKα2 in MCF-7 cells resulted in stronger p38MAPK activation in response to chemical AMPK activation or metabolic stress. Moreover, the same signaling was observed in HCC1500 cells, which endogenously express AMPKα2. Additionally, we cultured our cell lines as spheroids in order to mimic a tumor microenvironment. MCF-7 AMPKα2 cells formed larger, more viable spheres than control cells. In addition, the expression of AMPKα2 facilitated spheroid survival under hypoxic conditions. Finally, activation of p38MAPK was seen most abundantly in the MCF-7 AMPKα2 spheres. Our in vitro studies indicate an AMPKα2-dependent regulation of p38MAPK in response to metabolic stress in order to promote cancer cell survival. To evaluate cancer dormancy in vivo, MCF-7 cells expressing either GFP or AMPKα2 were injected into athymic nude mice previously implanted with slow-release estradiol pellets. After one week, the estradiol pellets were removed to induce cellular dormancy for thirty days. Analysis of tumors at this time indicated that more of the AMPKα2 expressing cells survived estradiol deprivation than did the control cells. Analysis of proliferation by Ki67 staining indicated that the GFP cells maintained proliferation during deprivation, while AMPKα2 cells were largely negative for proliferation. ApoTag staining revealed a similar trend for apoptotic cells. This suggests an inability to control cell cycle resulted in a decreased survival of the GFP cells under estradiol deprivation. Consistent with our observed in vitro cell signaling, AMPKα2 expressing tumors expressed higher levels of phospho-p38MAPK than GFP expressing tumors. Following the deprivation period, estradiol pellets were re-implanted and residual dormant tumors resumed growth. AMPKα2 tumors grew to roughly double the size of GFP tumors. Interestingly, AMPKα2 tumors had a higher number of mitotic events than did GFP tumors as visualized by Ki67 staining. This could be due to more viable cells being present following estradiol deprivation. We conclude that the expression of AMPKα2 promotes long-term breast cancer survival in estrogen-sensitive cells. Citation Format: Katie L. Sullivan, Stavros Kopsiaftis, Kathryn N. Phoenix, Melissa M. Fox, Kevin P. Claffey. AMPK promotes survival of breast cancer cells by modulating metabolic stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1138. doi:10.1158/1538-7445.AM2015-1138

Abstract 2661: Patient-derived breast cancer targeting heavy chain antibodies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The emergence of tumor-targeting antibodies such as Trastuzumab, Rituximab and anti-CD47 antibody has highlighted the significant role of antibodies for effective cancer therapy. The most common approach to generate tumor specific antibodies is to select an established tumor specific antigen and generate antibodies to that target. The drawback with this approach is that it does not lead to identification of novel antigens or antigenic epitopes. Therefore, we developed a strategy that will lead to the identification of both tumor specific antibodies and novel tumor antigens. The approach uses the patients immune system to guide us to define tumor specific antigens. In order to achieve this, we identified reactive germinal centers in the tumor draining sentinel lymph nodes from breast cancer patients. Immunohistochemical analysis of reactive germinal centers defined the B-cell regions for mRNA isolation and generation of cDNA molecules for the variable heavy chain segment (VH) of antibodies. The VH cDNA molecules were cloned into a mammalian expression vector to generate bivalent heavy chain antibodies with a C-terminus mouse Fc tag. At present, we have generated about 250 antigen-driven variable heavy chain clones from 27 breast cancer patient lymph node samples. We screened 46 heavy chain antibodies against a panel of breast cancer cell lines using immunofluorescence and identified 8 antibodies that showed strong and specific staining. Of these 8 antibodies, one of them (VH6-93) showed membrane staining preferentially on MDA-MB-231 cells in comparison to MCF7 cells. VH6-93 antibody also showed strong staining on conditionally reprogrammed tumor cells derived from a breast cancer patient with only moderate staining on normal breast epithelial cells derived from the same patient. VH6-93 also revealed preferential binding to breast tumor tissues as compared to normal ductal tissues evidenced by immunofluorescence and immunohistochemistry. To identify the target antigens, a multi-tiered approach was used, which included immunoprecipitation of target antigen from cell lysates followed by peptide sequencing with mass spectrometry as well as utilizing Nucleic Acid Programmable Protein Array (NAPPA) technology. A comprehensive analysis of two patient-derived antibodies has produced a panel of potential antigen targets, which are undergoing specificity and validation analysis. In summary, this methodology of using activated B-cells from sentinel lymph nodes is a unique way to enrich for tumor-specific antibody clones. The findings from this research will not only lead to tumor specific antibodies but will also reveal novel tumor specific antigens. Identification of these novel antigens can give rise to additional therapeutic strategies such as peptide or protein vaccination. Citation Format: Charan Kumar V. Devarakonda, Daniel Kita, Kathryn N. Phoenix, Dewey M. Magee, Kevin P. Claffey. Patient-derived breast cancer targeting heavy chain antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2014-2661