Electron microscopy imaging and mechanical characterization of T47D multicellular tumor spheroids-Older spheroids reduce interstitial space and become stiffer.

Multicellular cancer spheroids are an in vitro tissue model that mimics the three-dimensional microenvironment. As spheroids grow, they develop the gradients of oxygen, nutrients, and catabolites, affecting crucial tumor characteristics such as proliferation and treatment responses. The measurement of spheroid stiffness provides a quantitative measure to evaluate such structural changes over time. In this report, we measured the stiffness of size-matched day 5 and day 20 tumor spheroids using a custom-built microscale force sensor and conducted transmission electron microscopy (TEM) imaging to compare the internal structures. We found that older spheroids reduce interstitial spaces in the core region and became significantly stiffer. The measured elastic moduli were 260±100 and 680±150 Pa, for day 5 and day 20 spheroids, respectively. The day 20 spheroids showed an optically dark region in the center. Analyzing the high-resolution TEM images of spheroid middle sections across the diameter showed that the cells in the inner region of the day 20 spheroids are significantly larger and more closely packed than those in the outer regions. On the other hand, the day 5 spheroids did not show a significant difference between the inner and outer regions. The observed reduction of the interstitial space may be one factor that contributes to stiffer older spheroids.

PLCß2 Promotes VEGF-Induced Vascular Permeability.

BACKGROUND: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. METHODS: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) ß2. RESULTS: Global knock-out of PLCß2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCß2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCß2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCß2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. CONCLUSIONS: The results implicate PLCß2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCß2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

Label-Free Morphological Phenotyping of In Vitro 3D Microtumors.

By combining novel micro-scale manipulation cantilevers with commercially available, widely used 3D light microscopy, we were able to develop a new method of 3D elastography specialized for the analysis of 3D microtumors. Existing mechanical characterization methods are available for the study of single cells, using forces in the range of sub pN to a few hundred nN, or of larger tissues, with forces greater than 1 mN. Our method supports the mechanical analysis of micro- to meso-scale 3D tissues, such as multicellular spheroids (200-300 µm diameter), by applying forces in the range of sub-hundred nN to sub-mN, while also maintaining a spatial resolution of elasticity measurement as small as 20-30 µm. We use a differential interference contrast (DIC)/confocal microscope to obtain a 4D (x, y, z, and indentation steps) image sequence, which is then analyzed using our custom 3D pattern-tracking MATLAB program. With this method, we have been able to show structural and spatial heterogeneity among single cells and surrounding regions in tumor spheroids, and between different cell types in tumor-fibroblast co-cultured spheroids. Our method has the potential to both bridge the gap between in vitro monolayer culture systems and in vivo animal studies and add a mechanical component to existing biological assays.

GRK2 enforces androgen receptor dependence in the prostate and prostate tumors.

Metastatic tumors that have become resistant to androgen deprivation therapy represent the major challenge in treating prostate cancer. Although these recurrent tumors typically remain dependent on the androgen receptor (AR), non-AR-driven tumors that also emerge are particularly deadly and becoming more prevalent. Here, we present a new genetically engineered mouse model for non-AR-driven prostate cancer that centers on a negative regulator of G protein-coupled receptors that is downregulated in aggressive human prostate tumors. Thus, prostate-specific expression of a dominant-negative G protein-coupled receptor kinase 2 (GRK2-DN) transgene diminishes AR and AR target gene expression in the prostate, and confers resistance to castration-induced involution. Further, the GRK2-DN transgene dramatically accelerates oncogene-initiated prostate tumorigenesis by increasing primary tumor size, potentiating visceral organ metastasis, suppressing AR, and inducing neuroendocrine marker mRNAs. In summary, GRK2 enforces AR-dependence in the prostate, and the loss of GRK2 function in prostate tumors accelerates disease progression toward the deadliest stage.

Overcoming hypoxia-induced chemoresistance to cisplatin through tumor oxygenation monitored by optical imaging.

Perfluorocarbon nanoparticles have been reported to deliver oxygen to tumors and reduce hypoxia-induced radioresistance, however few studies have been carried out to study its role in reducing hypoxia-induced chemoresistance. The oxygenation effect also varies dramatically between different perfluorocarbon formulations and protocols, and there have been no efficient tools to monitor dynamic changes of tumor oxygenation non-invasively. Our goal was to promote tumor oxygenation using perfluorooctyl bromide (PFOB) nanoemulsion and to assess its role in sensitizing tumors to cisplatin treatment. A novel optical imaging protocol was also created to monitor the dynamic changes of tumor oxygenation in real-time. Methods: PFOB nanoemulsion with high oxygen-carrying capacity was prepared and administered to tumor-bearing mice intravenously. Tumor oxygenation was monitored using optical imaging with a hypoxia probe injected intratumorally, thus the oxygenation dynamics and best oxygenation protocol were determined. Various treatment groups were studied, and the tumor growth was monitored to evaluate the role of oxygenation in sensitizing tumors to cisplatin treatment. Results: PFOB nanoemulsion with and without pre-oxygenation along with carbogen breathing resulted in much better tumor oxygenation compared to carbogen breathing alone, while PFOB with air breathing did not show significant increase in tumor oxygenation. Pre-oxygenated PFOB with carbogen breathing produced the most effective oxygenation as early as 5 min post administration. In vitro and in vivo data showed preoxygenated PFOB nanoemulsion with carbogen breathing could increase cisplatin-mediated apoptosis of cancer cells and inhibited tumor growth at a low dose of cisplatin (1 mg/kg) treatment. Furthermore, the treatment did not induce nephrotoxicity. Conclusions: Preoxygenated PFOB nanoemulsion with carbogen breathing can effectively increase tumor oxygenation, which has a great potential to prevent/overcome hypoxia-induced chemotherapy resistance. In addition, optical imaging with intratumoral injection of the hypoxia probe was an efficient tool to monitor tumor oxygenation dynamics during PFOB administration, providing better understanding on oxygenation effects under different protocols.

Elastography of multicellular spheroids using 3D light microscopy.

We have demonstrated a new method of 3D elastography based on 3D light microscopy and micro-scale manipulation. We used custom-built micromanipulators to apply a mechanical force onto multicellular tumor spheroids (200-300 µm in size) and recorded the induced compression with a differential interference contrast (DIC)/confocal microscope to obtain a 4D (x, y, z, and indentation steps) image sequence. Deformation analysis made through 3D pattern tracking without using fluorescence revealed 3D structural and spatial heterogeneity in tumor spheroids. We observed a 20-30 µm-sized spot of locally-induced large deformation within a tumor spheroid. We also found solid fibroblast cores formed in a tumor-fibroblast co-culture spheroid to be stiffer than surrounding cancer cells, which would not have been discovered using only conventional fluorescence. Our new method of 3D elastography may be used to better understand structural composition in multicellular spheroids through analysis of mechanical heterogeneity.

Contribution of three-dimensional architecture and tumor-associated fibroblasts to hepcidin regulation in breast cancer.

Hepcidin is a peptide hormone that negatively regulates iron efflux and plays an important role in controlling the growth of breast tumors. In patients with breast cancer, the combined expression of hepcidin and its membrane target, ferroportin, predict disease outcome. However, mechanisms that control hepcidin expression in breast cancer cells remain largely unknown. Here, we use three-dimensional breast cancer spheroids derived from cell lines and breast cancer patients to probe mechanisms of hepcidin regulation in breast cancer. We observe that the extent of hepcidin induction and pathways of its regulation are markedly changed in breast cancer cells grown in three dimensions. In monolayer culture, BMPs, particularly BMP6, regulate hepcidin transcription. When breast cancer cells are grown as spheroids, there is a >10-fold induction in hepcidin transcripts. Microarray analysis combined with knockdown experiments reveal that GDF-15 is the primary mediator of this change. The increase in hepcidin as breast cells develop a three-dimensional architecture increases intracellular iron, as indicated by an increase in the iron storage protein ferritin. Immunohistochemical staining of human breast tumors confirms that both GDF-15 and hepcidin are expressed in breast cancer specimens. Further, levels of GDF-15 are significantly correlated with levels of hepcidin at both the mRNA and protein level in patient samples, consistent with a role for GDF-15 in control of hepcidin in human breast tumors. Inclusion of tumor-associated fibroblasts in breast cancer spheroids further induces hepcidin. This induction is mediated by fibroblast-dependent secretion of IL-6. Breast cancer cells grown as spheroids are uniquely receptive to IL-6-dependent induction of hepcidin by tumor-associated fibroblasts, since IL-6 does not induce hepcidin in cells grown as monolayers. Collectively, our results suggest a new paradigm for tumor-mediated control of iron through the control of hepcidin by tumor architecture and the breast tumor microenvironment.

A RAS-CaMKKß-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPß through a novel 3'UTR mechanism.

Oncogene-induced senescence (OIS) is an intrinsic tumor suppression mechanism that requires the p53 and RB pathways and post-translational activation of C/EBPß through the RAS-ERK cascade. We previously reported that in transformed/proliferating cells, C/EBPß activation is inhibited by G/U-rich elements (GREs) in its 3'UTR. This mechanism, termed "3'UTR regulation of protein activity" (UPA), maintains C/EBPß in a low-activity state in tumor cells and thus facilitates senescence bypass. Here we show that C/EBPß UPA is overridden by AMPK signaling. AMPK activators decrease cytoplasmic levels of the GRE binding protein HuR, which is a key UPA component. Reduced cytoplasmic HuR disrupts 3'UTR-mediated trafficking of Cebpb transcripts to the peripheral cytoplasm-a fundamental feature of UPA-thereby stimulating C/EBPß activation and growth arrest. In primary cells, oncogenic RAS triggers a Ca++-CaMKKß-AMPKα2-HuR pathway, independent of AMPKα1, that is essential for C/EBPß activation and OIS. This axis is disrupted in cancer cells through down-regulation of AMPKα2 and CaMKKß. Thus, CaMKKß-AMPKα2 signaling constitutes a key tumor suppressor pathway that activates a novel UPA-cancelling mechanism to unmask the cytostatic and pro-senescence functions of C/EBPß.

Stiffness analysis of 3D spheroids using microtweezers.

We describe a novel mechanical characterization method that has directly measured the stiffness of cancer spheroids for the first time to our knowledge. Stiffness is known to be a key parameter that characterizes cancerous and normal cells. Atomic force microscopy or optical tweezers have been typically used for characterization of single cells with the measurable forces ranging from sub pN to a few hundred nN, which are not suitable for measurement of larger 3D cellular structures such as spheroids, whose mechanical characteristics have not been fully studied. Here, we developed microtweezers that measure forces from sub hundred nN to mN. The wide force range was achieved by the use of replaceable cantilevers fabricated from SU8, and brass. The chopstick-like motion of the two cantilevers facilitates easy handling of samples and microscopic observation for mechanical characterization. The cantilever bending was optically tracked to find the applied force and sample stiffness. The efficacy of the method was demonstrated through stiffness measurement of agarose pillars with known concentrations. Following the initial system evaluation with agarose, two cancerous (T47D and BT474) and one normal epithelial (MCF 10A) breast cell lines were used to conduct multi-cellular spheroid measurements to find Young's moduli of 230, 420 and 1250 Pa for BT474, T47D, and MCF 10A, respectively. The results showed that BT474 and T47D spheroids are six and three times softer than epithelial MCF10A spheroids, respectively. Our method successfully characterized samples with wide range of Young's modulus including agarose (25-100 kPa), spheroids of cancerous and non-malignant cells (190-200 µm, 230-1250 Pa) and collagenase-treated spheroids (215 µm, 130 Pa).

AMPKα Is Suppressed in Bladder Cancer through Macrophage-Mediated Mechanisms.

Bladder cancer presents as either low- or high-grade disease, each with distinct mutational profiles; however, both display prominent mTORC1 activation. One major negative regulator of mTORC1 is AMPK, which is a critical metabolic regulator that suppresses cellular growth in response to metabolic stress by negatively regulating mTORC1. Alterations in the activation and protein levels of AMPK have been reported in breast, gastric, and hepatocellular carcinoma. To investigate whether AMPK suppression is responsible for mTOR activation in bladder cancer, the levels of AMPKα were quantified in a cohort of primary human bladder cancers and adjacent nontumor tissues. The levels of p-AMPKα, AMPKα1, AMPKα2, and total AMPKα were significantly suppressed in both low- and high-grade disease when compared with nontumor tissue. To elucidate the AMPKα suppression mechanism, we focused on inflammation, particularly tumor-infiltrating macrophages, due to their reported role in regulating AMPK expression. Treatment of HTB2 cancer cells with varying doses of differentiated U937 macrophage conditioned medium (CM) demonstrated a dose-dependent reduction of AMPKα protein. Additionally, macrophage CM treatment of HTB2 and HT1376 bladder cells for various times also reduced AMPKα protein but not mRNA levels. Direct TNFα treatment also suppressed AMPKα at the protein but not RNA level. Finally, staining of the human cohort for CD68, a macrophage marker, revealed that CD68+ cell counts correlated with reduced AMPKα levels. In summary, these data demonstrate the potential role for inflammation and inflammatory cytokines in regulating the levels of AMPKα and promoting mTORC1 activation in bladder cancer.

AMPKα2 Regulates Bladder Cancer Growth through SKP2-Mediated Degradation of p27.

AMP-activated protein kinase (AMPK) is the central metabolic regulator of the cell and controls energy consumption based upon nutrient availability. Due to its role in energy regulation, AMPK has been implicated as a barrier for cancer progression and is suppressed in multiple cancers. To examine whether AMPK regulates bladder cancer cell growth, HTB2 and HT1376 bladder cells were treated with an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). AICAR treatment reduced proliferation and induced the expression of p27Kip1 (CDKN1B), which was mediated through an mTOR-dependent mechanism. Interestingly, AMPKα2 knockdown resulted in reduced p27 levels, whereas AMPKα1 suppression did not. To further determine the exact mechanism by which AMPKa2 regulates p27, HTB2 and HT1376 cells were transduced with an shRNA targeting AMPKα2. Stable knockdown of AMPKα2 resulted in increased proliferation and decreased p27 protein. The reduced p27 protein was determined to be dependent upon SKP2. Additionally, loss of AMPKα2 in a xenograft and a chemical carcinogen model of bladder cancer resulted in larger tumors with less p27 protein and high SKP2 levels. Consistent with the regulation observed in the bladder cancer model systems, a comprehensive survey of human primary bladder cancer clinical specimens revealed low levels of AMPKα2 and p27 and high levels of SKP2. IMPLICATIONS: These results highlight the contribution of AMPKα2 as a mechanism for controlling bladder cancer growth by regulating proliferation through mTOR suppression and induction of p27 protein levels, thus indicating how AMPKα2 loss may contribute to tumorigenesis. Mol Cancer Res; 14(12); 1182-94. ©2016 AACR.

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

BACKGROUND: Monoclonal 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. METHODS: Eight 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. RESULTS: Flow 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. CONCLUSIONS: From 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.

Inhibition of breast cancer cell migration by activation of cAMP signaling.

Almost all deaths from breast cancer arise from metastasis of the transformed cells to other sites in the body. Hence, uncovering a means of inhibiting breast cancer cell migration would provide a significant advance in the treatment of this disease. Stimulation of the cAMP signaling pathway has been shown to inhibit migration and motility of a number of cell types. A very effective way of selectively stimulating cAMP signaling is through inhibition of cyclic nucleotide phosphodiesterases (PDEs). Therefore, we examined full expression profiles of all known PDE genes at the mRNA and protein levels in four human breast cancer cell lines and eight patients' breast cancer tissues. By these analyses, expression of almost all PDE genes was seen in both cell lines and tissues. In the cell lines, appreciable expression was seen for PDEs 1C, 2A, 3B, 4A, 4B, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 9A, 10A, and 11A. In patients' tissues, appreciable expression was seen for PDEs 1A, 3B, 4A, 4B, 4C, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 8B, and 9A. PDE8A mRNA in particular is prominently expressed in all cell lines and patients' tissue samples examined. We show here that stimulation of cAMP signaling with cAMP analogs, forskolin, and PDE inhibitors, including selective inhibitors of PDE3, PDE4, PDE7, and PDE8, inhibit aggressive triple negative MDA-MB-231 breast cancer cell migration. Under the same conditions, these agents had little effect on breast cancer cell proliferation. This study demonstrates that PDE inhibitors inhibit breast cancer cell migration, and thus may be valuable therapeutic targets for inhibition of breast cancer metastasis. Since PDE8A is expressed in all breast cancer samples, and since dipyridamole, which inhibits PDE8, and PF-04957325, a selective PDE8 inhibitor, both inhibit migration, it suggests that PDE8A may be a valuable novel target for treatment of this disease.

Cardiomyopathy and Worsened Ischemic Heart Failure in SM22-α Cre-Mediated Neuropilin-1 Null Mice: Dysregulation of PGC1α and Mitochondrial Homeostasis.

OBJECTIVE: Neuropilin-1 (NRP-1) is a multidomain membrane receptor involved in angiogenesis and development of neuronal circuits, however, the role of NRP-1 in cardiovascular pathophysiology remains elusive. APPROACH AND RESULTS: In this study, we first observed that deletion of NRP-1 induced peroxisome proliferator-activated receptor γ coactivator 1α in cardiomyocytes and vascular smooth muscle cells, which was accompanied by dysregulated cardiac mitochondrial accumulation and induction of cardiac hypertrophy- and stress-related markers. To investigate the role of NRP-1 in vivo, we generated mice lacking Nrp-1 in cardiomyocytes and vascular smooth muscle cells (SM22-α-Nrp-1 KO), which exhibited decreased survival rates, developed cardiomyopathy, and aggravated ischemia-induced heart failure. Mechanistically, we found that NRP-1 specifically controls peroxisome proliferator-activated receptor γ coactivator 1 α and peroxisome proliferator-activated receptor γ in cardiomyocytes through crosstalk with Notch1 and Smad2 signaling pathways, respectively. Moreover, SM22-α-Nrp-1 KO mice exhibited impaired physical activities and altered metabolite levels in serum, liver, and adipose tissues, as demonstrated by global metabolic profiling analysis. CONCLUSIONS: Our findings provide new insights into the cardioprotective role of NRP-1 and its influence on global metabolism.

Increased expression of L-selectin (CD62L) in high-grade urothelial carcinoma: A potential marker for metastatic disease.

INTRODUCTION: L-Selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leukocytes with a primary function of directing leukocyte migration and homing of lymphocytes to lymph nodes. In a gene expression microarray study comparing laser-captured microdissected high-grade muscle-invasive bladder cancer (MIBC) without prior treatment and low-grade bladder cancer (LGBC) human samples, we found CD62L to be the highest differentially expressed gene. We sought to examine the differential expression of CD62L in MIBCs and its clinical relevance. METHODS: Unfixed fresh and formalin-fixed paraffin-embedded human bladder cancer specimens and serum samples were obtained from the University of Connecticut Health Center tumor bank. Tumor cells were isolated from frozen tumor tissue sections by laser-captured microdissected followed by RNA isolation. Quantitative polymerase chain reaction was used to validate the level of CD62L transcripts. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to evaluate the CD62L protein localization and expression level. Flow cytometry was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine database. RESULTS: Immunostaining showed a uniformly higher expression of CD62L in MIBC specimens vs. LGBCs specimens. Further, CD62L localization was seen in foci of metastatic tumor cells in lymph node specimens from patients with high-grade MIBC and known nodal involvement. Up-regulated expression of CD62L was also observed by flow cytometric analysis of freshly isolated tumor cells from biopsies of high-grade cancers vs. LGBC specimens. Circulating CD62L levels were also found to be higher in serum samples from patients with high-grade metastatic vs. high-grade nonmetastatic MIBC. In addition, in silico analysis of Oncomine Microarray Database showed a significant correlation between CD62L expression and tumor aggressiveness and clinical outcomes. CONCLUSION: These data confirm the expression of CD62L on urothelial carcinoma cells and suggest that CD62L may serve as biomarker to predict the presence of or risk for developing metastatic disease in patients with bladder cancer.

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.

Expression of phosphodiesterase 6 (PDE6) in human breast cancer cells.

Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In this study we examined established human breast cancer cell lines and patients' primary breast cancer tissues for expression of genetic components of phosphodiesterase 6 (PDE6), a cGMP-specific PDE involved in transduction of the light signal, and previously thought to be selectively expressed in photoreceptors. By microarray analysis we find highly significant expression of mRNA for the PDE6B, PDE6C, and PDE6D genes in both the cell lines and patients' tissues, minimal expression of PDE6A and PDE6G and no expression of PDE6H. Using antibody specific for PDE6ß, we find expression of PDE6B protein in a wide range of patients' tissues by immunohistochemistry, and in MCF-7 breast cancer cells by immunofluorescence and Western blot analysis. Considerable expression of key circadian genes, PERIOD 2, CLOCK, TIMELESS, CRYPTOCHROME 1, and CRYPTOCHROME 2 was also seen in all breast cancer cell lines and all patients' breast cancer tissues. These studies indicate that genes for PDE6 and control of circadian rhythm are expressed in human breast cancer cells and tissues and may play a role in transducing the effects of light on 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.

Prostate specific membrane antigen (PSMA) regulates angiogenesis independently of VEGF during ocular neovascularization.

BACKGROUND: Aberrant growth of blood vessels in the eye forms the basis of many incapacitating diseases and currently the majority of patients respond to anti-angiogenic therapies based on blocking the principal angiogenic growth factor, vascular endothelial growth factor (VEGF). While highly successful, new therapeutic targets are critical for the increasing number of individuals susceptible to retina-related pathologies in our increasingly aging population. Prostate specific membrane antigen (PSMA) is a cell surface peptidase that is absent on normal tissue vasculature but is highly expressed on the neovasculature of most solid tumors, where we have previously shown to regulate angiogenic endothelial cell invasion. Because pathologic angiogenic responses are often triggered by distinct signals, we sought to determine if PSMA also contributes to the pathologic angiogenesis provoked by hypoxia of the retina, which underlies many debilitating retinopathies. METHODOLOGY/PRINCIPAL FINDINGS: Using a mouse model of oxygen-induced retinopathy, we found that while developmental angiogenesis is normal in PSMA null mice, hypoxic challenge resulted in decreased retinal vascular pathology when compared to wild type mice as assessed by avascular area and numbers of vascular tufts/glomeruli. The vessels formed in the PSMA null mice were more organized and highly perfused, suggesting a more 'normal' phenotype. Importantly, the decrease in angiogenesis was not due to an impaired hypoxic response as levels of pro-angiogenic factors are comparable; indicating that PSMA regulation of angiogenesis is independent of VEGF. Furthermore, both systemic and intravitreal administration of a PSMA inhibitor in wild type mice undergoing OIR mimicked the PSMA null phenotype resulting in improved retinal vasculature. CONCLUSIONS/SIGNIFICANCE: Our data indicate that PSMA plays a VEGF-independent role in retinal angiogenesis and that the lack of or inhibition of PSMA may represent a novel therapeutic strategy for treatment of angiogenesis-based ocular diseases.