Abstract We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor ...leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis.
Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as ...addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method using ARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays.
Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of ...context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.
Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by ...developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.
The initial step of metastasis in carcinomas, often referred to as the epithelial-mesenchymal transition (EMT), occurs via the loss of adherens junctions (e.g. cadherins) by the tumor embolus. This ...leads to a subsequent loss of cell polarity and cellular differentiation and organization, enabling cells of the embolus to become motile and invasive. However highly malignant inflammatory breast cancer (IBC) over-expresses E-cadherin. The human xenograft model of IBC (MARY-X), like IBC, displays the signature phenotype of an exaggerated degree of lymphovascular invasion (LVI) in situ by tumor emboli. An intact E-cadherin/alpha, beta-catenin axis mediates the tight, compact clump of cells found both in vitro and in vivo as spheroids and tumor emboli, respectively.
Using electron microscopy and focused ion beam milling to acquire in situ sections, we performed ultrastructural analysis of both an IBC and non-IBC, E-cadherin positive cell line to determine if retention of this adhesion molecule contributed to cellular organization.
Here we report through ultrastructural analysis that IBC exhibits a high degree of cellular organization with polar elements such as apical/lateral positioning of E-cadherin, apical surface microvilli, and tortuous lumen-like (canalis) structures. In contrast, agarose-induced spheroids of MCF-7, a weakly invasive E-cadherin positive breast carcinoma cell line, do not exhibit ultrastructural polar features.
This study has determined that the highly metastatic IBC with an exaggerated malignant phenotype challenges conventional wisdom in that instead of displaying a loss of cellular organization, IBC acquires a highly structured architecture.These findings suggest that the metastatic efficiency might be linked to the formation and maintenance of these architectural features. The comparative architectural features of both the spheroid and embolus of MARY-X provide an in vitro model with tractable in vivo applications.
The study of extracellular vesicles (EVs) in cancer progression is a complex and rapidly evolving field. Whole categories of cellular interactions in cancer which were originally presumed to be due ...solely to soluble secreted molecules have now evolved to include membrane-enclosed extracellular vesicles (EVs), which include both exosomes and shed microvesicles (MVs), and can contain many of the same molecules as those secreted in soluble form but many different molecules as well. EVs released by cancer cells can transfer mRNA, miRNA, and proteins to different recipient cells within the tumor microenvironment, in both an autocrine and paracrine manner, causing a significant impact on signaling pathways, mRNA transcription, and protein expression. The transfer of EVs to target cells, in turn, supports cancer growth, immunosuppression, and metastasis formation. This review focuses exclusively on breast cancer EVs with an emphasis on breast cancer-derived exosomes, keeping in mind that breast cancer-derived EVs share some common physical properties with EVs of other cancers.
Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity ...properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.
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•Pathologic protein networks and their engagement in clinic are monitored by imaging•Real-time tumor pharmacometric data are obtained at the level of individual tumors•Theranostic and clinical assay combined provide quantitative tumor measurements•The platform provides dose and schedule information for epichaperome targeting
Pillarsetty et al. demonstrate the ability to visualize the epichaperome, a pathologic protein-protein interaction network, and to measure inhibitor engagement from mice and patients at single-tumor resolution in real time, which facilitates the optimization of dose and schedule selection.
The limited translational value in clinic of analyses performed on 2-D cell cultures has prompted a shift toward the generation of 3-dimensional (3-D) multicellular systems. Here we present a ...spontaneously-forming in vitro cancer spheroid model, referred to as spheroids(MARY-X), that precisely reflects the pathophysiological features commonly found in tumor tissues and the lymphovascular embolus. In addition, we have developed a rapid, inexpensive means to evaluate response following drug treatment where spheroid dissolution indices from brightfield image analyses are used to construct dose-response curves resulting in relevant IC50 values. Using the spheroids(MARY-X) model, we demonstrate the unique ability of a new class of molecules, containing the caged Garcinia xanthone (CGX) motif, to induce spheroidal dissolution and apoptosis at IC50 values of 0.42 +/-0.02 μM for gambogic acid and 0.66 +/-0.02 μM for MAD28. On the other hand, treatment of spheroids(MARY-X) with various currently approved chemotherapeutics of solid and blood-borne cancer types failed to induce any response as indicated by high dissolution indices and subsequent poor IC50 values, such as 7.8 +/-3.1 μM for paclitaxel. Our studies highlight the significance of the spheroids(MARY-X) model in drug screening and underscore the potential of the CGX motif as a promising anticancer pharmacophore.
Inflammatory breast cancer (IBC) is the most lethal form of breast cancers with a 5-year survival rate of only 40 %. Despite its lethality, IBC remains poorly understood which has greatly limited its ...therapeutic management. We thus decided to utilize an integrative functional genomic strategy to identify the Achilles' heel of IBC cells.
We have pioneered the development of genetic tools as well as experimental and analytical strategies to perform RNAi-based loss-of-function studies at a genome-wide level. Importantly, we and others have demonstrated that these functional screens are able to identify essential functions linked to certain cancer phenotypes. Thus, we decided to use this approach to identify IBC specific sensitivities.
We identified and validated HDAC6 as a functionally necessary gene to maintain IBC cell viability, while being non-essential for other breast cancer subtypes. Importantly, small molecule inhibitors for HDAC6 already exist and are in clinical trials for other tumor types. We thus demonstrated that Ricolinostat (ACY1215), a leading HDAC6 inhibitor, efficiently controls IBC cell proliferation both in vitro and in vivo. Critically, functional HDAC6 dependency is not associated with genomic alterations at its locus and thus represents a non-oncogene addiction. Despite HDAC6 not being overexpressed, we found that its activity is significantly higher in IBC compared to non-IBC cells, suggesting a possible rationale supporting the observed dependency.
Our finding that IBC cells are sensitive to HDAC6 inhibition provides a foundation to rapidly develop novel, efficient, and well-tolerated targeted therapy strategies for IBC patients.
After the publication of this article 1 the authors noticed two small mistakes in the affiliations listings 1. 1) Dr. Ruth Rodriguez should be marked as an equal contributor alongside Dr. Putcha and ...Dr. Yu. 2. 2) Dr. Ruth Rodriguez’s correct affiliation is ‘Department of Pathology, Icahn School of Medicine at Mount Sinai , New York, NY 10029-6574, USA’ and not ‘Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA’ as the original article states.
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