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.

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.

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.

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.

Revealing the role of phospholipase Cß3 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 Cß3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCß3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.

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.

Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression in mice.

Tumor vasculature is hyperpermeable to macromolecules compared to normal vasculature; however, the relationship between tumor hyperpermeability and tumor progression is poorly understood. Here we show that a cell-permeable peptide derived from caveolin-1, termed cavtratin, reduces microvascular hyperpermeability and delays tumor progression in mice. These antipermeability and antitumor actions of cavtratin occur in the absence of direct cytostatic or antiangiogenic effects. Cavtratin blocks microvascular permeability by inhibiting endothelial nitric oxide synthase (eNOS), as the antipermeability and antitumor actions of cavtratin are markedly diminished in eNOS knockout mice. Our results support the concepts that hyperpermeability of tumor blood vessels contributes to tumor progression and that blockade of eNOS may be exploited as a novel target for antitumor therapy.

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.

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.

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.

Argonaute-2 expression is regulated by epidermal growth factor receptor and mitogen-activated protein kinase signaling and correlates with a transformed phenotype in breast cancer cells.

Argonaute (Ago) 2 is the catalytic engine of mammalian RNA interference, but little is known concerning the regulation of Ago2 by cell-signaling pathways. In this study we show that expression of Ago2, but not Ago1, Ago3, or Ago4, is elevated in estrogen receptor (ER) alpha-negative (ERalpha(-)) vs. ERalpha-positive (ERalpha+) breast cancer cell lines, and in ERalpha(-) breast tumors. In MCF-7 cells the low level of Ago2 was found to be dependent upon active ERalpha/estrogen signaling. Interestingly, the high expression of Ago2 in ERalpha(-) cells was severely blunted by inhibition of the epidermal growth factor (EGF) receptor/MAPK signaling pathway, using either a pharmacological MAPK kinase inhibitor, U0126, or a small interfering RNA directed against EGF receptor. Half-life studies using cycloheximide indicated that EGF enhanced, whereas U0126 decreased, Ago2 protein stability. Furthermore, a proteosome inhibitor, MG132, blocked Ago2 protein turnover. The functional consequences of elevated Ago2 levels were examined by stable transfection of ERalpha+ MCF-7 cells with full-length and truncated forms of Ago2. The full-length Ago2 transfectants displayed enhanced proliferation, reduced cell-cell adhesion, and increased migratory ability, as shown by proliferation, homotypic aggregation, and wound healing assays, respectively. Overexpression of full-length Ago2, but not truncated forms of Ago2 or an empty vector control, reduced the levels of E-cadherin, beta-catenin, and beta-actin, as well as enhanced endogenous miR-206 activity. These data indicate that Ago2 is regulated at both the transcriptional and posttranslational level, and also implicate Ago2 and enhanced micro-RNA activity in the tumorigenic progression of breast cancer cell lines.

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.

Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERalpha negative MDA-MB-435 breast cancer model.

Metformin, a first line treatment for type 2 diabetes, has been implicated as a potential anti-neoplastic agent for breast cancers as well as other cancers. Metformin is known to work in part through the activation of AMP-dependent kinase (AMPK). AMPK is a key regulator of cellular energy homeostasis, especially under stress conditions where biosynthetic pathways are blocked by the phosphorylation of downstream AMPK substrates. Stimulation of AMPK by metformin resulted in a significant repression of cell proliferation and active MAPK1/2 in both estrogen receptor alpha (ERalpha) negative (MDA-MB-231, MDA-MB-435) and positive (MCF-7, T47D) human breast cancer cell lines. However, when ERalpha negative MDA-MB-435 cells were treated with metformin, they demonstrated increased expression of vascular endothelial growth factor (VEGF) in an AMPK dependent manner; while the ERalpha positive MCF-7 cells did not. Systemic therapy with metformin was tested for efficacy in an orthotopic model of ERalpha negative breast cancer performed in athymic nude mice. Surprisingly, metformin therapy significantly improved tumorigenic progression as compared to untreated controls. The metformin-treated group showed increased VEGF expression, intratumoral microvascular density and reduced necrosis. Metformin treatment was sufficient, however, to reduce systemic IGF-1 and the proliferation rate of tumor cells in vascularized regions. The data presented here suggests that, although metformin significantly represses breast cancer cell growth in vitro, the efficacy with respect to its therapeutic application for ERalpha negative breast cancer lesions in vivo may result in promotion of the angiogenic phenotype and increased tumorigenic progression.

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.

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.

Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1.

CONTEXT: Progesterone (P4) influences ovarian cancer cells by an unknown mechanism. OBJECTIVE: The objective was to determine whether P4 acts through progesterone receptor membrane component-1 (PGRMC1) in ovarian cancers. DESIGN, SETTING AND PATIENTS: Archival tissue and cDNA provided by OriGene were used for expression studies. In vitro experiments were conducted with Ovcar-3 cells. MAIN OUTCOME MEASURES: PCR, Western blot, and immunohistochemistry were used to measure expression of PGRMC1 and nuclear progesterone receptor (PGR). PGRMC1's role in regulating the viability of ovarian cancers was assessed by overexpressing PGRMC1, depleting PGRMC1 using small interfering RNA, and attenuating PGRMC1's action with a blocking antibody. Apoptosis was determined by 4',6'-diamino-2-phenylindole staining. RESULTS: PGRMC1 mRNA increased and PGR mRNA decreased in advanced stages of ovarian cancer. Unlike PGR, PGRMC1 was expressed in virtually every cancer cell within the tumor. A similar relationship between PGRMC1 and PGR was observed in Ovcar-3 cells. In these cells P4 suppressed apoptosis induced by either serum withdrawal or cisplatin (CDDP). Moreover, in the presence of P4, the following occurs: 1) overexpression of PGRMC1 reduces the effectiveness of CDDP, 2) depletion of PGRMC1 with small interfering RNA enhances the effects of CDDP, and 3) PGRMC1 antibody treatment increases the apoptotic response to CDDP. CONCLUSIONS: These findings indicate that PGRMC1 plays an important role in promoting ovarian cancer cell viability and that attenuating PGRMC1's action makes the ovarian cancer cells more sensitive to CDDP. These data suggest that targeted depletion of PGRMC1 could be useful as an adjunct to CDDP therapy.

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).