Ang II acutely stimulates Na,K-pump in cells from proximal tubules by increasing its phosphorylation at S938 via a PI3K/AKT pathway.

Angiotensin II (Ang II)-dependent stimulation of the AT1 receptor in proximal tubules increases sodium reabsorption and blood pressure. Reabsorption is driven by the Na,K-pump that is acutely stimulated by Ang II, which requires phosphorylation of serine-938 (S938). This site is present in humans and only known to phosphorylated by PKA. Yet, activation of AT1 decreases cAMP required to activate PKA and inhibiting PKA does not block Ang II-dependent phosphorylation of S938. We tested the hypothesis that Ang II-dependent activation is mediated via increased phosphorylation at S938 through a PI3K/AKT-dependent pathway. Experiments were conducted using opossum kidney cells, a proximal tubule cell line, stably co-expressing the AT1 receptor and either the wild-type (α-1.wild-type) or an alanine substituted (α-1.S938A) form of rat kidney Na,K-pump. A 5-min exposure to 10 pM Ang II significantly activated Na,K-pump activity (56%) measured as short-circuit current across polarized α-1.wild-type cells. Wortmannin, at a concentration that selectively inhibits PI3K, blocked that Ang II-dependent activation. Ang II did not stimulate Na,K-pump activity in α-1.S938A cells. Ang II at 10 and 100 pM increased phosphorylation at S938 in α-1.wild-type cells measured in whole cell lysates. The increase was inhibited by wortmannin plus H-89, an inhibitor of PKA, not by either alone. Ang II activated AKT inhibited by wortmannin, not H-89. These data support our hypothesis and show that Ang II-dependent phosphorylation at S938 stimulates Na,K-pump activity and transcellular sodium transport.

Modeling Tumor: Lymphatic Interactions in Lymphatic Metastasis of Triple Negative Breast Cancer.

Breast cancer frequently metastasizes to lymphatics and the presence of breast cancer cells in regional lymph nodes is an important prognostic factor. Delineating the mechanisms by which breast cancer cells disseminate and spatiotemporal aspects of interactions between breast cancer cells and lymphatics is needed to design new therapies to prevent lymphatic metastases. As triple-negative breast cancer (TNBC) has a high incidence of lymphatic metastasis, we used a three-dimensional (3D) coculture model of human TNBC cells and human microvascular lymphatic endothelial cells (LECs) to analyze TNBC:LEC interactions. Non-invasive analyses such as live-cell imaging in real-time and collection of conditioned media for secretomic analysis were facilitated by our novel microfluidic chambers. The volumes of 3D structures formed in TNBC:LEC cocultures are greater than that of 3D structures formed by either LEC or TNBC monocultures. Over 4 days of culture there is an increase in multicellular invasive outgrowths from TNBC spheroids and an association of TNBC spheroids with LEC networks. The increase in invasive phenotype also occurred when TNBC spheroids were cultured in LEC-conditioned media and in wells linked to ones containing LEC networks. Our results suggest that modeling spatiotemporal interactions between TNBC and LECs may reveal paracrine signaling that could be targeted to reduce lymphatic metastasis.

Anti-tumor and immune modulating activity of T cell induced tumor-targeting effectors (TITE).

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) showed promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occurs upon engagement with tumor cells via the bispecific antibody (BiAb) bridge, which stimulates BATs to release cytotoxic molecules, cytokines, chemokines, and other signaling molecules extracellularly. We hypothesized that the release of BATs Induced Tumor-Targeting Effectors (TITE) by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune-activating immunotherapeutic approach. In a 3D tumorsphere model, TITE showed potent cytotoxic activity against multiple breast cancer cell lines compared to control conditioned media (CM): Tumor-CM (T-CM), BATs-CM (B-CM), BiAb Armed PBMC-CM (BAP-CM) or PBMC-CM (P-CM). Multiplex cytokine analysis showed high levels of Th1 cytokines and chemokines; phospho-protein signaling array data suggest that the prominent JAK1/STAT1 pathway may be responsible for the induction and release of Th1 cytokines/chemokines in TITE. In xenograft breast cancer models, IV injections of 10× concentrated TITE (3×/week for 3 weeks; 150 µl TITE/injection) was able to inhibit tumor growth significantly (ICR/scid, p < 0.003; NSG p < 0.008) compared to the control mice. We tested the key components of the TITE for immune activating and anti-tumor activity individually and in combinations, the combination of IFN-γ, TNF-α and MIP-1ß recapitulates the key activities of the TITE. In summary, master mix of active components of BATs-Tumor complex-derived TITE can provide a clinically controllable cell-free platform to target various tumor types regardless of the heterogeneous nature of the tumor cells and mutational tumor.

Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer.

Cells grown in three dimensions (3D) within natural extracellular matrices or synthetic scaffolds more closely recapitulate the phenotype of those cells within tissues in regard to normal developmental and pathobiological processes. This includes degradation of the surrounding stroma as the cells migrate and invade through the matrices. As 3D cultures of tumor cells predict efficacy of, and resistance to, a wide variety of cancer therapies, we employed tissue-engineering approaches to establish 3D pathomimetic avatars of human breast cancer cells alone and in the context of both their cellular and pathochemical microenvironments. We have shown that we can localize and quantify key parameters of malignant progression by live-cell imaging of the 3D avatars over time (4D). One surrogate for changes in malignant progression is matrix degradation, which can be localized and quantified by our live-cell proteolysis assay. This assay is predictive of changes in spatio-temporal and dynamic interactions among the co-cultured cells and changes in viability, proliferation, and malignant phenotype. Furthermore, our live-cell proteolysis assay measures the effect of small-molecule inhibitors of proteases and kinases, neutralizing or blocking antibodies to cytokines and photodynamic therapy on malignant progression. We suggest that 3D/4D pathomimetic avatars in combination with our live-cell proteolysis assays will be a useful preclinical screening platform for cancer therapies. Our ultimate goal is to develop 3D/4D avatars from an individual patient's cancer in which we can screen "personalized medicine" therapies using changes in proteolytic activity to quantify therapeutic efficacy.

Breast Cancer: Proteolysis and Migration.

Understanding breast cancer cell proteolysis and migration is crucial for developing novel therapies to prevent local and distant metastases. Human cancer cells utilize many biological functions comparable to those observed during embryogenesis conferring the cancer cells with survival advantages. One such advantage is the ability to secrete proteases into the tumor microenvironment in order to remodel the extracellular matrix to facilitate migration. These proteases degrade the extracellular matrix, which initially functions as a barrier to cancer cell escape from their site of origin. The extracellular matrix also functions as a reservoir for growth factors that can be released by the secreted proteases and thereby further aid tumor growth and progression. Other survival advantages of tumor cells include: the ability to utilize multiple modes of motility, thrive in acidic microenvironments, and the tumor cell's ability to hijack stromal and immune cells to foster their own migration and survival. In order to reduce metastasis, we must focus our efforts on addressing the survival advantages that tumor cells have acquired.

Acidosis and proteolysis in the tumor microenvironment.

The glycolytic phenotype of the Warburg effect is associated with acidification of the tumor microenvironment. In this review, we describe how acidification of the tumor microenvironment may increase the invasive and degradative phenotype of cancer cells. As a template of an extracellular acidic microenvironment that is linked to proteolysis, we use the resorptive pit formed between osteoclasts and bone. We describe similar changes that have been observed in cancer cells in response to an acidic microenvironment and that are associated with proteolysis and invasive and metastatic phenotypes. This includes consideration of changes observed in the intracellular trafficking of vesicles, i.e., lysosomes and exosomes, and in specialized regions of the membrane, i.e., invadopodia and caveolae. Cancer-associated cells are known to affect what is generally referred to as tumor proteolysis but little direct evidence for this being regulated by acidosis; we describe potential links that should be verified.

Ras and Rap1: A tale of two GTPases.

Ras oncoproteins play pivotal roles in both the development and maintenance of many tumor types. Unfortunately, these proteins are difficult to directly target using traditional pharmacological strategies, in part due to their lack of obvious binding pockets or allosteric sites. This obstacle has driven a considerable amount of research into pursuing alternative ways to effectively inhibit Ras, examples of which include inducing mislocalization to prevent Ras maturation and inactivating downstream proteins in Ras-driven signaling pathways. Ras proteins are archetypes of a superfamily of small GTPases that play specific roles in the regulation of many cellular processes, including vesicle trafficking, nuclear transport, cytoskeletal rearrangement, and cell cycle progression. Several other superfamily members have also been linked to the control of normal and cancer cell growth and survival. For example, Rap1 has high sequence similarity to Ras, has overlapping binding partners, and has been demonstrated to both oppose and mimic Ras-driven cancer phenotypes. Rap1 plays an important role in cell adhesion and integrin function in a variety of cell types. Mechanistically, Ras and Rap1 cooperate to initiate and sustain ERK signaling, which is activated in many malignancies and is the target of successful therapeutics. Here we review the role activated Rap1 in ERK signaling and other downstream pathways to promote invasion and cell migration and metastasis in various cancer types.

In Vitro Models for Studying Invasive Transitions of Ductal Carcinoma In Situ.

About one fourth of all newly identified cases of breast carcinoma are diagnoses of breast ductal carcinoma in situ (DCIS). Since we cannot yet distinguish DCIS cases that would remain indolent from those that may progress to life-threatening invasive ductal carcinoma (IDC), almost all women undergo aggressive treatment. In order to allow for more rational individualized treatment, we and others are developing in vitro models to identify and validate druggable pathways that mediate the transition of DCIS to IDC. These models range from conventional two-dimensional (2D) monolayer cultures on plastic to 3D cultures in natural or synthetic matrices. Some models consist solely of DCIS cells, either cell lines or primary cells. Others are co-cultures that include additional cell types present in the normal or cancerous human breast. The 3D co-culture models more accurately mimic structural and functional changes in breast architecture that accompany the transition of DCIS to IDC. Mechanistic studies of the dynamic and temporal changes associated with this transition are facilitated by adapting the in vitro models to engineered microfluidic platforms. Ultimately, the goal is to create in vitro models that can serve as a reproducible preclinical screen for testing therapeutic strategies that will reduce progression of DCIS to IDC. This review will discuss the in vitro models that are currently available, as well as the progress that has been made using them to understand DCIS pathobiology.

Downregulation of Rap1Gap: A Switch from DCIS to Invasive Breast Carcinoma via ERK/MAPK Activation.

Diagnosis of breast ductal carcinoma in situ (DCIS) presents a challenge since we cannot yet distinguish those cases that would remain indolent and not require aggressive treatment from cases that may progress to invasive ductal cancer (IDC). The purpose of this study is to determine the role of Rap1Gap, a GTPase activating protein, in the progression from DCIS to IDC. Immunohistochemistry (IHC) analysis of samples from breast cancer patients shows an increase in Rap1Gap expression in DCIS compared to normal breast tissue and IDCs. In order to study the mechanisms of malignant progression, we employed an in vitro three-dimensional (3D) model that more accurately recapitulates both structural and functional cues of breast tissue. Immunoblotting results show that Rap1Gap levels in MCF10.Ca1D cells (a model of invasive carcinoma) are reduced compared to those in MCF10.DCIS (a model of DCIS). Retroviral silencing of Rap1Gap in MCF10.DCIS cells activated extracellular regulated kinase (ERK) mitogen-activated protein kinase (MAPK), induced extensive cytoskeletal reorganization and acquisition of mesenchymal phenotype, and enhanced invasion. Enforced reexpression of Rap1Gap in MCF10.DCIS-Rap1GapshRNA cells reduced Rap1 activity and reversed the mesenchymal phenotype. Similarly, introduction of dominant negative Rap1A mutant (Rap1A-N17) in DCIS-Rap1Gap shRNA cells caused a reversion to nonmalignant phenotype. Conversely, expression of constitutively active Rap1A mutant (Rap1A-V12) in noninvasive MCF10.DCIS cells led to phenotypic changes that were reminiscent of Rap1Gap knockdown. Thus, reduction of Rap1Gap in DCIS is a potential switch for progression to an invasive phenotype. The Graphical Abstract summarizes these findings.

Pathomimetic avatars reveal divergent roles of microenvironment in invasive transition of ductal carcinoma in situ.

BACKGROUND: The breast tumor microenvironment regulates progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). However, it is unclear how interactions between breast epithelial and stromal cells can drive this progression and whether there are reliable microenvironmental biomarkers to predict transition of DCIS to IDC. METHODS: We used xenograft mouse models and a 3D pathomimetic model termed mammary architecture and microenvironment engineering (MAME) to study the interplay between human breast myoepithelial cells (MEPs) and cancer-associated fibroblasts (CAFs) on DCIS progression. RESULTS: Our results show that MEPs suppress tumor formation by DCIS cells in vivo even in the presence of CAFs. In the in vitro MAME model, MEPs reduce the size of 3D DCIS structures and their degradation of extracellular matrix. We further show that the tumor-suppressive effects of MEPs on DCIS are linked to inhibition of urokinase plasminogen activator (uPA)/urokinase plasminogen activator receptor (uPAR)-mediated proteolysis by plasminogen activator inhibitor 1 (PAI-1) and that they can lessen the tumor-promoting effects of CAFs by attenuating interleukin 6 (IL-6) signaling pathways. CONCLUSIONS: Our studies using MAME are, to our knowledge, the first to demonstrate a divergent interplay between MEPs and CAFs within the DCIS tumor microenvironment. We show that the tumor-suppressive actions of MEPs are mediated by PAI-1, uPA and its receptor, uPAR, and are sustained even in the presence of the CAFs, which themselves enhance DCIS tumorigenesis via IL-6 signaling. Identifying tumor microenvironmental regulators of DCIS progression will be critical for defining a robust and predictive molecular signature for clinical use.

Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches.

Methodologies to image and quantify the activity of proteolytic enzymes have been developed in an effort to identify protease-related druggable pathways that are involved in malignant progression of cancer. Our laboratory has pioneered techniques for functional live-cell imaging of protease activity in pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by tumor cells in 3-D cultures over time (4D). In order to recapitulate the cellular composition and architecture of tumors in the pathomimetic avatars, we include other tumor-associated cells (e.g., fibroblasts, myoepithelial cells, microvascular endothelial cells). We also model noncellular aspects of the tumor microenvironment such as acidic pericellular pH. Use of pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify, and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for screening of therapeutic modalities, we have designed and fabricated custom 3D culture chambers with multiple wells that are either individual or connected by a channel to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. We are using the pathomimetic avatars to identify druggable pathways, screen drug and natural product libraries and accelerate entry of validated drugs or natural products into clinical trials.

Pathomimetic cancer avatars for live-cell imaging of protease activity.

Proteases are essential for normal physiology as well as multiple diseases, e.g., playing a causative role in cancer progression, including in tumor angiogenesis, invasion, and metastasis. Identification of dynamic alterations in protease activity may allow us to detect early stage cancers and to assess the efficacy of anti-cancer therapies. Despite the clinical importance of proteases in cancer progression, their functional roles individually and within the context of complex protease networks have not yet been well defined. These gaps in our understanding might be addressed with: 1) accurate and sensitive tools and methods to directly identify changes in protease activities in live cells, and 2) pathomimetic avatars for cancer that recapitulate in vitro the tumor in the context of its cellular and non-cellular microenvironment. Such avatars should be designed to facilitate mechanistic studies that can be translated to animal models and ultimately the clinic. Here, we will describe basic principles and recent applications of live-cell imaging for identification of active proteases. The avatars optimized by our laboratory are three-dimensional (3D) human breast cancer models in a matrix of reconstituted basement membrane (rBM). They are designated mammary architecture and microenvironment engineering (MAME) models as they have been designed to mimic the structural and functional interactions among cell types in the normal and cancerous human breast. We have demonstrated the usefulness of these pathomimetic avatars for following dynamic and temporal changes in cell:cell interactions and quantifying changes in protease activity associated with these interactions in real-time (4D). We also briefly describe adaptation of the avatars to custom-designed and fabricated tissue architecture and microenvironment engineering (TAME) chambers that enhance our ability to analyze concomitant changes in the malignant phenotype and the associated tumor microenvironment.

How to Target Activated Ras Proteins: Direct Inhibition vs. Induced Mislocalization.

Oncogenic Ras proteins are a driving force in a significant set of human cancers and wildtype, unmutated Ras proteins likely contribute to the malignant phenotype of many more. The overall challenge of targeting activated Ras proteins has great promise to treat cancer, but this goal has yet to be achieved. Significant efforts and resources have been committed to inhibiting Ras, but these energies have so far made little impact in the clinic. Direct attempts to target activated Ras proteins have faced many obstacles, including the fundamental nature of the gain-of-function oncogenic activity being produced by a loss-of-function at the biochemical level. Nevertheless, there has been very promising recent pre-clinical progress. The major strategy that has so far reached the clinic aimed to inhibit activated Ras indirectly through blocking its post-translational modification and inducing its mislocalization. While these efforts to indirectly target Ras through inhibition of farnesyl transferase (FTase) were rationally designed, this strategy suffered from insufficient attention to the distinctions between the isoforms of Ras. This led to subsequent failures in large-scale clinical trials targeting K-Ras driven lung, colon, and pancreatic cancers. Despite these setbacks, efforts to indirectly target activated Ras through inducing its mislocalization have persisted. It is plausible that FTase inhibitors may still have some utility in the clinic, perhaps in combination with statins or other agents. Alternative approaches for inducing mislocalization of Ras through disruption of its palmitoylation cycle or interaction with chaperone proteins are in early stages of development.

Dynamic microglial modulation of spatial learning and social behavior.

Microglia are active players in inflammation, but also have important supporting roles in CNS maintenance and function, including modulation of neuronal activity. We previously observed an increase in the frequency of excitatory postsynaptic current in organotypic brain slices after depletion of microglia using clodronate. Here, we describe that local hippocampal depletion of microglia by clodronate alters performance in tests of spatial memory and sociability. Global depletion of microglia by high-dose oral administration of a Csf1R inhibitor transiently altered spatial memory but produced no change in sociability behavior. Microglia depletion and behavior effects were both reversible, consistent with a dynamic role for microglia in the regulation of such behaviors.

The experimental autoimmune encephalomyelitis disease course is modulated by nicotine and other cigarette smoke components.

Epidemiological studies have reported that cigarette smoking increases the risk of developing multiple sclerosis (MS) and accelerates its progression. However, the molecular mechanisms underlying these effects remain unsettled. We have investigated here the effects of the nicotine and the non-nicotine components in cigarette smoke on MS using the experimental autoimmune encephalomyelitis (EAE) model, and have explored their underlying mechanism of action. Our results show that nicotine ameliorates the severity of EAE, as shown by reduced demyelination, increased body weight, and attenuated microglial activation. Nicotine administration after the development of EAE symptoms prevented further disease exacerbation, suggesting that it might be useful as an EAE/MS therapeutic. In contrast, the remaining components of cigarette smoke, delivered as cigarette smoke condensate (CSC), accelerated and increased adverse clinical symptoms during the early stages of EAE, and we identify a particular cigarette smoke compound, acrolein, as one of the potential mediators. We also show that the mechanisms underlying the opposing effects of nicotine and CSC on EAE are likely due to distinct effects on microglial viability, activation, and function.

Biomimetic tissue-engineered systems for advancing cancer research: NCI Strategic Workshop report.

Advanced technologies and biomaterials developed for tissue engineering and regenerative medicine present tractable biomimetic systems with potential applications for cancer research. Recently, the National Cancer Institute convened a Strategic Workshop to explore the use of tissue biomanufacturing for development of dynamic, physiologically relevant in vitro and ex vivo biomimetic systems to study cancer biology and drug efficacy. The workshop provided a forum to identify current progress, research gaps, and necessary steps to advance the field. Opportunities discussed included development of tumor biomimetic systems with an emphasis on reproducibility and validation of new biomimetic tumor models, as described in this report.

p53 opens the mitochondrial permeability transition pore to trigger necrosis.

Ischemia-associated oxidative damage leading to necrosis is a major cause of catastrophic tissue loss, and elucidating its signaling mechanism is therefore of paramount importance. p53 is a central stress sensor responding to multiple insults, including oxidative stress to orchestrate apoptotic and autophagic cell death. Whether p53 can also activate oxidative stress-induced necrosis is, however, unknown. Here, we uncover a role for p53 in activating necrosis. In response to oxidative stress, p53 accumulates in the mitochondrial matrix and triggers mitochondrial permeability transition pore (PTP) opening and necrosis by physical interaction with the PTP regulator cyclophilin D (CypD). Intriguingly, a robust p53-CypD complex forms during brain ischemia/reperfusion injury. In contrast, reduction of p53 levels or cyclosporine A pretreatment of mice prevents this complex and is associated with effective stroke protection. Our study identifies the mitochondrial p53-CypD axis as an important contributor to oxidative stress-induced necrosis and implicates this axis in stroke pathology.

Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury.

BACKGROUND: Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. METHODS AND FINDINGS: Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH(+) and Iba-1(+) cells acutely died, and as the penumbra the area surrounding the core where Iba-1(+) cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. CONCLUSIONS: Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic.