Redistribution of the glycocalyx exposes phagocytic determinants on apoptotic cells.

Phagocytes remove dead and dying cells by engaging "eat-me" ligands such as phosphatidylserine (PtdSer) on the surface of apoptotic targets. However, PtdSer is obscured by the bulky exofacial glycocalyx, which also exposes ligands that activate "don't-eat-me" receptors such as Siglecs. Clearly, unshielding the juxtamembrane "eat-me" ligands is required for the successful engulfment of apoptotic cells, but the mechanisms underlying this process have not been described. Using human and murine cells, we find that apoptosis-induced retraction and weakening of the cytoskeleton that anchors transmembrane proteins cause an inhomogeneous redistribution of the glycocalyx: actin-depleted blebs emerge, lacking the glycocalyx, while the rest of the apoptotic cell body retains sufficient actin to tether the glycocalyx in place. Thus, apoptotic blebs can be engaged by phagocytes and are targeted for engulfment. Therefore, in cells with an elaborate glycocalyx, such as mucinous cancer cells, this "don't-come-close-to-me" barrier must be removed to enable clearance by phagocytosis.

A tumor-restricted glycoform of podocalyxin is a highly selective marker of immunologically cold high-grade serous ovarian carcinoma.

Introduction: Targeted-immunotherapies such as antibody-drug conjugates (ADC), chimeric antigen receptor (CAR) T cells or bispecific T-cell engagers (eg, BiTE®) all aim to improve cancer treatment by directly targeting cancer cells while sparing healthy tissues. Success of these therapies requires tumor antigens that are abundantly expressed and, ideally, tumor specific. The CD34-related stem cell sialomucin, podocalyxin (PODXL), is a promising target as it is overexpressed on a variety of tumor types and its expression is consistently linked to poor prognosis. However, PODXL is also expressed in healthy tissues including kidney podocytes and endothelia. To circumvent this potential pitfall, we developed an antibody, named PODO447, that selectively targets a tumor-associated glycoform of PODXL. This tumor glycoepitope is expressed by 65% of high-grade serous ovarian carcinoma (HGSOC) tumors. Methods: In this study we characterize these PODO447-expressing tumors as a distinct subset of HGSOC using four different patient cohorts that include pre-chemotherapy, post-neoadjuvant chemotherapy (NACT) and relapsing tumors as well as tumors from various peritoneal locations. Results: We find that the PODO447 epitope expression is similar across tumor locations and negligibly impacted by chemotherapy. Invariably, tumors with high levels of the PODO447 epitope lack infiltrating CD8+ T cells and CD20+ B cells/plasma cells, an immune phenotype consistently associated with poor outcome. Discussion: We conclude that the PODO447 glycoepitope is an excellent biomarker of immune "cold" tumors and a candidate for the development of targeted-therapies for these hard-to-treat cancers.

Antibody-Drug Conjugates Targeting Tumor-Specific Mucin Glycoepitopes.

Finding the ideal epitope to target is a key element for the development of an antibody-drug conjugate (ADC). To maximize drug delivery to tumor cells and reduce side effects, this epitope should be specific to cancer cells and spare all normal tissue. During cancer progression, glycosylation pathways are frequently altered leading to the generation of new glycosylation patterns selective to cancer cells. Mucins are highly glycosylated proteins frequently expressed on tumors and, thus, ideal presenters of altered glycoepitopes. In this review, we describe three different types of glycoepitopes that are recognized by monoclonal antibodies (mAb) and, therefore, serve as ideal scaffolds for ADC; glycan-only, glycopeptide and shielded-peptide glycoepitopes. We review pre-clinical and clinical results obtained with ADCs targeting glycoepitopes expressed on MUC1 or podocalyxin (Podxl) and two mAbs targeting glycoepitopes expressed on MUC16 or MUC5AC as potential candidates for ADC development. Finally, we discuss current limits in using glycoepitope-targeting ADCs to treat cancer and propose methods to improve their efficacy and specificity.

Filamin A regulates caspase-3 cleavage in platelets in a protein kinase C (PKC)-dependent manner.

Apoptosis is a critical process for the maintenance of cell populations, and involves mitochondrial depolarization, the sequential cleavage of caspase-9 and -3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskeleton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopying the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specifically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus providing an improved understanding of how circulating platelet counts are maintained.

Modeling Collective Invasion and Single-Cell Mesenchymal Invasion in Three-Dimensional Matrigel-Collagen I Cultures.

Understanding the modes and mechanisms of tumor cell invasion is key to developing targeted therapies against metastatic disease. In vitro assays modeling tumor progression have primarily been optimized for studying classical single-cell migration through an epithelial-mesenchymal transition (EMT). Although experimental and clinical histopathological evidence has revealed that tumor invasion is plastic and that epithelial carcinomas can invade by a range of modes that vary from single, mesenchyme-like cells, all the way to cohesive, collective units, few in vitro assays have been designed to assess these modes specifically. Thus, we have developed a Matrigel-Collagen I overlay assay that is suitable for identifying and quantifying both collective and mesenchymal invasion. This three-dimensional (3D) culture assay utilizes the features of Matrigel and Collagen I to mimic the laminin-rich basement membrane and the stiff, fibrillar Collagen I tumor microenvironment allowing for spheroid invasion to be assessed at the interface between these two matrix components.

Targeting a Tumor-Specific Epitope on Podocalyxin Increases Survival in Human Tumor Preclinical Models.

Podocalyxin (Podxl) is a CD34-related cell surface sialomucin that is normally highly expressed by adult vascular endothelia and kidney podocytes where it plays a key role in blocking adhesion. Importantly, it is also frequently upregulated on a wide array of human tumors and its expression often correlates with poor prognosis. We previously showed that, in xenograft studies, Podxl plays a key role in metastatic disease by making tumor initiating cells more mobile and invasive. Recently, we developed a novel antibody, PODO447, which shows exquisite specificity for a tumor-restricted glycoform of Podxl but does not react with Podxl expressed by normal adult tissue. Here we utilized an array of glycosylation defective cell lines to further define the PODO447 reactive epitope and reveal it as an O-linked core 1 glycan presented in the context of the Podxl peptide backbone. Further, we show that when coupled to monomethyl auristatin E (MMAE) toxic payload, PODO447 functions as a highly specific and effective antibody drug conjugate (ADC) in killing ovarian, pancreatic, glioblastoma and leukemia cell lines in vitro. Finally, we demonstrate PODO447-ADCs are highly effective in targeting human pancreatic and ovarian tumors in xenografted NSG and Nude mouse models. These data reveal PODO447-ADCs as exquisitely tumor-specific and highly efficacious immunotherapeutic reagents for the targeting of human tumors. Thus, PODO447 exhibits the appropriate characteristics for further development as a targeted clinical immunotherapy.

Inflammation-Induced Metastatic Colonization of the Lung Is Facilitated by Hepatocyte Growth Factor-Secreting Monocyte-Derived Macrophages.

A rate-limiting step for circulating tumor cells to colonize distant organ sites is their ability to locate a microenvironmental niche that supports their survival and growth. This can be achieved by features intrinsic to the tumor cells and/or by the conditioning of a "premetastatic" niche. To determine if pulmonary inflammation promotes the latter, we initiated models for inflammatory asthma, hypersensitivity pneumonitis, or bleomycin-induced sterile inflammation before introducing tumor cells with low metastatic potential into the circulation. All types of inflammation increased the end-stage metastatic burden of the lungs 14 days after tumor cell inoculation without overtly affecting tumor extravasation. Instead, the number and size of early micrometastatic lesions found within the interstitial tissues 96 hours after tumor cell inoculation were increased in the inflamed lungs, coincident with increased tumor cell survival and the presence of nearby inflammation-induced monocyte-derived macrophages (MoDM; CD11b+CD11c+). Remarkably, the adoptive transfer of these MoDM was sufficient to increase lung metastasis in the absence of inflammation. These inflammation-induced MoDM secrete a number of growth factors and cytokines, one of which is hepatocyte growth factor (HGF), that augmented tumor cell survival under conditions of stress in vitro. Importantly, blocking HGF signaling with the cMET inhibitor capmatinib abolished inflammation-induced early micrometastatic lesion formation in vivo. These findings indicate that inflammation-induced MoDM and HGF in particular increase the efficiency of early metastatic colonization in the lung by locally preconditioning the microenvironment. IMPLICATIONS: Inflammation preconditions the distant site microenvironment to increase the metastatic potential of tumor cells that arrive there.

MAPS: machine-assisted phenotype scoring enables rapid functional assessment of genetic variants by high-content microscopy.

BACKGROUND: Genetic testing is widely used in evaluating a patient's predisposition to hereditary diseases. In the case of cancer, when a functionally impactful mutation (i.e. genetic variant) is identified in a disease-relevant gene, the patient is at elevated risk of developing a lesion in their lifetime. Unfortunately, as the rate and coverage of genetic testing has accelerated, our ability to assess the functional status of new variants has fallen behind. Therefore, there is an urgent need for more practical, streamlined and cost-effective methods for classifying variants. RESULTS: To directly address this issue, we designed a new approach that uses alterations in protein subcellular localization as a key indicator of loss of function. Thus, new variants can be rapidly functionalized using high-content microscopy (HCM). To facilitate the analysis of the large amounts of imaging data, we developed a new software toolkit, named MAPS for machine-assisted phenotype scoring, that utilizes deep learning to extract and classify cell-level features. MAPS helps users leverage cloud-based deep learning services that are easy to train and deploy to fit their specific experimental conditions. Model training is code-free and can be done with limited training images. Thus, MAPS allows cell biologists to easily incorporate deep learning into their image analysis pipeline. We demonstrated an effective variant functionalization workflow that integrates HCM and MAPS to assess missense variants of PTEN, a tumor suppressor that is frequently mutated in hereditary and somatic cancers. CONCLUSIONS: This paper presents a new way to rapidly assess variant function using cloud deep learning. Since most tumor suppressors have well-defined subcellular localizations, our approach could be widely applied to functionalize variants of uncertain significance and help improve the utility of genetic testing.

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin.

BACKGROUND: The success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is associated with poor outcome, thus emerging as an important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft models, Podxl expression promotes tumor growth and metastasis. Although a promising target for immunotherapy, the expression of Podxl on normal vascular endothelia and kidney podocytes could hamper efforts to therapeutically target this molecule. Since pathways regulating post-translational modifications are frequently perturbed in cancer cells, we sought to produce novel anti-Podxl antibodies (Abs) that selectively recognize tumor-restricted glycoepitopes on the extracellular mucin domain of Podxl. METHODS: Splenic B cells were isolated from rabbits immunized with a Podxl-expressing human tumor cell line. Abs from these B cells were screened for potent reactivity to Podxl+ neoplastic cell lines but not Podxl+ primary endothelial cells. Transcripts encoding heavy and light chain variable regions from promising B cells were cloned and expressed as recombinant proteins. Tumor specificity was assessed using primary normal tissue and an ovarian cancer tissue microarray (TMA). Mapping of the tumor-restricted epitope was performed using enzyme-treated human tumor cell lines and a glycan array. RESULTS: One mAb (PODO447) showed strong reactivity with a variety of Podxl+ tumor cell lines but not with normal primary human tissue including Podxl+ kidney podocytes and most vascular endothelia. Screening of an ovarian carcinoma TMA (219 cases) revealed PODO447 reactivity with the majority of tumors, including 65% of the high-grade serous histotype. Subsequent biochemical analyses determined that PODO447 reacts with a highly unusual terminal N-acetylgalactosamine beta-1 (GalNAcß1) motif predominantly found on the Podxl protein core. Finally, Ab-drug conjugates showed specific efficacy in killing tumor cells in vitro. CONCLUSIONS: We have generated a novel and exquisitely tumor-restricted mAb, PODO447, that recognizes a glycoepitope on Podxl expressed at high levels by a variety of tumors including the majority of life-threatening high-grade serous ovarian tumors. Thus, tumor-restricted PODO447 exhibits the appropriate specificity for further development as a targeted immunotherapy.

Distinct Functional Requirements for Podocalyxin in Immature and Mature Podocytes Reveal Mechanisms of Human Kidney Disease.

Dominant and recessive mutations in podocalyxin (PODXL) are associated with human kidney disease. Interestingly, some PODXL mutations manifest as anuria while others are associated with proteinuric kidney disease. PODXL heterozygosity is associated with adult-onset kidney disease and podocalyxin shedding into the urine is a common biomarker of a variety nephrotic syndromes. It is unknown, however, how various lesions in PODXL contribute to these disparate disease pathologies. Here we generated two mouse stains: one that deletes Podxl in developmentally mature podocytes (Podxl∆Pod) and a second that is heterozygous for podocalyxin in all tissues (Podxl+/-). We used histologic and ultrastructural analyses, as well as clinical chemistry assays to evaluate kidney development and function in these strains. In contrast to null knockout mice (Podxl-/-), which die shortly after birth from anuria and hypertension, Podxl∆Pod mice develop an acute congenital nephrotic syndrome characterized by focal segmental glomerulosclerosis (FSGS) and proteinuria. Podxl+/- mice, in contrast, have a normal lifespan, and fail to develop kidney disease under normal conditions. Intriguingly, although wild-type C57Bl/6 mice are resistant to puromycin aminonucleoside (PA)-induced nephrosis (PAN), Podxl+/- mice are highly sensitive and PA induces severe proteinuria and collapsing FSGS. In summary, we find that the developmental timepoint at which podocalyxin is ablated (immature vs. mature podocytes) has a profound effect on the urinary phenotype due to its critical roles in both the formation and the maintenance of podocyte ultrastructure. In addition, Podxl∆Pod and Podxl+/- mice offer powerful new mouse models to evaluate early biomarkers of proteinuric kidney disease and to test novel therapeutics.

A Premalignant Cell-Based Model for Functionalization and Classification of PTEN Variants.

As sequencing becomes more economical, we are identifying sequence variations in the population faster than ever. For disease-associated genes, it is imperative that we differentiate a sequence variant as either benign or pathogenic, such that the appropriate therapeutic interventions or surveillance can be implemented. PTEN is a frequently mutated tumor suppressor that has been linked to the PTEN hamartoma tumor syndrome. Although the domain structure of PTEN and the functional impact of a number of its most common tumor-linked mutations have been characterized, there is a lack of information about many recently identified clinical variants. To address this challenge, we developed a cell-based assay that utilizes a premalignant phenotype of normal mammary epithelial cells lacking PTEN. We measured the ability of PTEN variants to rescue the spheroid formation phenotype of PTEN-/- MCF10A cells maintained in suspension. As proof of concept, we functionalized 47 missense variants using this assay, only 19 of which have clear classifications in ClinVar. We utilized a machine learning model trained with annotated genotypic data to classify variants as benign or pathogenic based on our functional scores. Our model predicted with high accuracy that loss of PTEN function was indicative of pathogenicity. We also determined that the pathogenicity of certain variants may have arisen from reduced stability of the protein product. Overall, this assay outperformed computational predictions, was scalable, and had a short run time, serving as an ideal alternative for annotating the clinical significance of cancer-associated PTEN variants. SIGNIFICANCE: Combined three-dimensional tumor spheroid modeling and machine learning classifies PTEN missense variants, over 70% of which are currently listed as variants of uncertain significance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2775/F1.large.jpg.

A sticky wicket: Defining molecular functions for CD34 in hematopoietic cells.

The CD34 cell surface antigen is widely expressed in tissues on cells with progenitor-like properties and on mature vascular endothelia. In adult human bone marrow, CD34 marks hematopoietic stem and progenitor cells (HSPCs) starting from the bulk of hematopoietic stem cells with long-term repopulating potential (LT-HSCs) throughout expansion and differentiation of oligopotent and unipotent progenitors. CD34 protein surface expression is typically lost as cells mature into terminal effectors. Because of this expression pattern of HSPCs, CD34 has had a central role in the evaluation or selection of donor graft tissue in HSC transplant (HSCT). Given its clinical importance, it is surprising that the biological functions of CD34 are still poorly understood. This enigma is due, in part, to CD34's context-specific role as both a pro-adhesive and anti-adhesive molecule and its potential functional redundancy with other sialomucins. Moreover, there are also critical differences in the regulation of CD34 expression on HSPCs in humans and experimental mice. In this review, we highlight some of the more well-defined functions of CD34 in HSPCs with a focus on proposed functions most relevant to HSCT biology.

CD44 Loss Disrupts Lung Lipid Surfactant Homeostasis and Exacerbates Oxidized Lipid-Induced Lung Inflammation.

Alveolar macrophages (AMs) are CD44 expressing cells that reside in the alveolar space where they maintain lung homeostasis by serving critical roles in immunosurveillance and lipid surfactant catabolism. AMs lacking CD44 are unable to bind the glycosaminoglycan, hyaluronan, which compromises their survival and leads to reduced numbers of AMs in the lung. Using RNA sequencing, lipidomics and multiparameter flow cytometry, we demonstrate that CD44-/- mice have impaired AM lipid homeostasis and increased surfactant lipids in the lung. CD44-/- AMs had increased expression of CD36, a lipid scavenger receptor, as well as increased intracellular lipid droplets, giving them a foamy appearance. RNA sequencing revealed the differential expression of genes associated with lipid efflux and metabolism in CD44-/- AMs. Lipidomic analysis showed increased lipids in both the supernatant and cell pellet extracted from the bronchoalveolar lavage of CD44-/- mice. Phosphatidylcholine species, cholesterol, oxidized phospholipids and levels of reactive oxygen species (ROS) were increased in CD44-/- AMs. Oxidized phospholipids were more cytotoxic to CD44-/- AMs and induced greater lung inflammation in CD44-/- mice. Reconstitution of CD44+/+ mice with CD44-/- bone marrow as well as adoptive transfer of CD44-/- AMs into CD44+/+ mice showed that lipid accumulation in CD44-/- AMs occurred irrespective of the lung environment, suggesting a cell intrinsic defect. Administration of colony stimulating factor 2 (CSF-2), a critical factor in AM development and maintenance, increased AM numbers in CD44-/- mice and decreased phosphatidylcholine levels in the bronchoalveolar lavage, but was unable to decrease intracellular lipid accumulation in CD44-/- AMs. Peroxisome proliferator-activated receptor gamma (PPARγ), downstream of CSF-2 signaling and a regulator of lipid metabolism, was reduced in the nucleus of CD44-/- AMs, and PPARγ inhibition in normal AMs increased their lipid droplets. Thus, CD44 deficiency causes defects in AMs that lead to abnormal lipid accumulation and oxidation, which exacerbates oxidized lipid-induced lung inflammation. Collectively, these findings implicate CD44 as a regulator of lung homeostasis and inflammation.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor.

Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tension-induced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated.

Loukoumasomes Are Distinct Subcellular Structures from Rods and Rings and Are Structurally Associated with MAP2 and the Nuclear Envelope in Retinal Cells.

"Rods and rings" (RR) and loukoumasomes are similarly shaped, subcellular macromolecular structures with as yet unknown function. RR, so named because of their shape, are formed in response to inhibition in the GTP or CTP synthetic pathways and are highly enriched in the two key enzymes of the nucleotide synthetic pathway. Loukoumasomes also occur as linear and toroidal bodies and were initially inferred to be the same as RR, largely due to their shared shape and size and the fact that it was unclear if they shared the same subcomponents. In human retinoblastoma tissue and cells we have observed toroidal, perinuclear, macromolecular structures of similar size and antigenicity to those previously reported in neurons (neuronal-loukoumasomes). To further characterize the subcomponents of the retinal-loukoumasomes, confocal analysis following immunocytochemical staining for alpha-tubulin, beta-III tubulin and detyrosinated tubulin was performed. These studies indicate that retinal-loukoumasomes are enriched for beta-III tubulin and other tubulins associated with microtubules. Immunofluorescence together with the in situ proximity ligation assay (PLA), confirmed that beta-III tubulin colocalized with detyrosinated tubulin within loukoumasomes. Our results indicate that these tissues contain only loukoumasomes because these macromolecular structures are immunoreactive with an anti-tubulin antibody but are not recognized by the prototype anti-RR/inosine monophosphate dehydrogenase (IMPDH) antibody (It2006). To further compare the RR and retinal-loukoumasomes, retinoblastoma cells were exposed to the IMPDH-inhibitor ribavirin, a drug known to induce the formation of RR. In contrast to RR, the production of retinal-loukoumasomes was unaffected. Coimmunostaining of Y79 cells for beta-III tubulin and IMPDH indicate that these cells, when treated with ribavirin, can contain both retinal-loukoumasomes and RR and that these structures are antigenically distinct. Subcellular fractionation studies indicate that ribavirin increased the RR subcomponent, IMPDH, in the nuclear fraction of Y79 cells from 21.3 ± 5.8% (0 mM ribavirin) to 122.8 ± 7.9% (1 mM ribavirin) while the subcellular localization of the retinal-loukoumasome subcomponent tubulin went unaltered. Further characterization of retinal-loukoumasomes in retinoblastoma cells reveals that they are intimately associated with lamin folds within the nuclear envelope. Using immunofluorescence and the in situ PLA in this cell type, we have observed colocalization of beta-III tubulin with MAP2. As MAP2 is a microtubule-associated protein implicated in microtubule crosslinking, this supports a role for microtubule crosslinkers in the formation of retinal-loukoumasomes. Together, these results suggest that loukoumasomes and RR are distinct subcellular macromolecular structures, formed by different cellular processes and that there are other loukoumasome-like structures within retinal tissues and cells.

Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C-COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain complexes required for their proliferation.

The cell surface mucin podocalyxin regulates collective breast tumor budding.

BACKGROUND: Overexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression. METHODS: MCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. RESULTS: Forced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture. CONCLUSIONS: Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.

Integrin-linked kinase regulates senescence in an Rb-dependent manner in cancer cell lines.

Anti-integrin-linked kinase (ILK) therapies result in aberrant mitosis including altered mitotic spindle organization, centrosome declustering and mitotic arrest. In contrast to cells that expressed the retinoblastoma tumor suppressor protein Rb, we have shown that in retinoblastoma cell lines that do not express Rb, anti-ILK therapies induced aberrant mitosis that led to the accumulation of temporarily viable multinucleated cells. The present work was undertaken to: 1) determine the ultimate fate of cells that had survived anti-ILK therapies and 2) determine whether or not Rb expression altered the outcome of these cells. Our data indicate that ILK, a chemotherapy drug target is expressed in both well-differentiated, Rb-negative and relatively undifferentiated, Rb-positive retinoblastoma tissue. We show that small molecule targeting of ILK in Rb-positive and Rb-deficient cancer cells results in increased centrosomal declustering, aberrant mitotic spindle formation and multinucleation. However, anti-ILK therapies in vitro have different outcomes in retinoblastoma and glioblastoma cell lines that depend on Rb expression. TUNEL labeling and propidium iodide FACS analysis indicate that Rb-positive cells exposed to anti-ILK therapies are more susceptible to apoptosis and senescence than their Rb-deficient counterparts wherein aberrant mitosis induced by anti-ILK therapies exhibit mitotic arrest instead. These studies are the first to show a role for ILK in chemotherapy-induced senescence in Rb-positive cancer lines. Taken together these results indicate that the oncosuppressive outcomes for anti-ILK therapies in vitro, depend on the expression of the tumor suppressor Rb, a known G1 checkpoint and senescence regulator.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

INTRODUCTION: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. METHODS: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. RESULTS: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. CONCLUSIONS: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.