Genomic instability and metabolism in cancer.

Genomic instability and metabolic reprogramming are among the key hallmarks discriminating cancer cells from normal cells. The two phenomena contribute to the robust and evasive nature of cancer, particularly when cancer cells are exposed to chemotherapeutic agents. Genomic instability is defined as the increased frequency of mutations within the genome, while metabolic reprogramming is the alteration of metabolic pathways that cancer cells undergo to adapt to increased bioenergetic demand. An underlying source of these mutations is the aggregate product of damage to the DNA, and a defective repair pathway, both resulting in the expansion of genomic lesions prior to uncontrolled proliferation and survival of cancer cells. Exploitation of DNA damage and the subsequent DNA damage response (DDR) have aided in defining therapeutic approaches in cancer. Studies have demonstrated that targeting metabolic reprograming yields increased sensitivity to chemo- and radiotherapies. In the past decade, it has been shown that these two key features are interrelated. Metabolism impacts DNA damage and DDR via regulation of metabolite pools. Conversely, DDR affects the response of metabolic pathways to therapeutic agents. Because of the interplay between genomic instability and metabolic reprogramming, we have compiled findings which more selectively highlight the dialog between metabolism and DDR, with a particular focus on glucose metabolism and double-strand break (DSB) repair pathways. Decoding this dialog will provide significant clues for developing combination cancer therapies.

Genomic instability and mitochondrial homeostasis in cancer: does chromatin have a say?

While genomic instability and mitochondrial homeostasis are integral for cancer progression, how these two hallmarks interact remains poorly understood. Here, we reflect on the dialogue between chromatin-based genomic instability and impairment of mitochondrial function and depict the importance of this interaction in cancer progression to metastasis.

The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44.

Hyaluronan accumulation in the tumor microenvironment is associated with poor prognosis in several solid human cancers. To understand the role of stromal hyaluronan in tumor progression, we engineered 3T3HAS3, a hyaluronan-producing fibroblast cell line, by lentiviral transduction of Balb/c 3T3 cells with the human hyaluronan synthase 3 (HAS3) gene. 3T3HAS3 cells significantly enhanced tumor growth when co-grafted with MDA-MB-468 cells in nude mice. Immunohistochemical analysis of the xenograft tumors showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of MDA-MB-468 + 3T3HAS3 co-grafts was greatly reduced upon hyaluronan degradation by lentiviral transduction of a human hyaluronidase gene in 3T3HAS3 cells, or by systemic administration of pegvorhyaluronidase alfa (PEGPH20). In contrast, the growth of the co-graft tumors was not inhibited when CD44 expression was reduced or ablated by small hairpin RNA-mediated CD44 knockdown in MDA-MB-468 cells, CD44 CRISPR knockout in 3T3HAS3 cells, or by grafting these cells in CD44 knockout nude mice. Collectively, these data demonstrate that tumor growth of an engineered xenograft breast cancer model with hyaluronan-accumulating stroma can be dependent on hyaluronan and independent of CD44.

Remodeling the Tumor Microenvironment Sensitizes Breast Tumors to Anti-Programmed Death-Ligand 1 Immunotherapy.

Immunotherapies targeting immune checkpoint inhibitors have changed the landscape of cancer treatment, however, many patients are resistant or refractory to immunotherapy. The sensitivity of tumor cells to immunotherapy may be influenced by hyaluronan (HA) accumulation in the tumor microenvironment (TME). Enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA) remodels the TME. This leads to reduced tumor interstitial pressure and decompressed tumor blood vessels, which are both associated with increased exposure of tumor cells to chemotherapy drugs. Here, we demonstrate PVHA increased the uptake of anti-programmed death-ligand 1 (PD-L1) antibody in HA-accumulating animal models of breast cancer. The increased levels of anti-PD-L1 antibody were associated with increased accumulation of T cells and natural killer cells and decreased myeloid-derived suppressor cells. PD-L1 blockade significantly inhibited tumor growth when combined with PVHA, but not alone. Our results suggest that PVHA can sensitize HA-accumulating tumors to anti-PD-L1 immunotherapy. SIGNIFICANCE: These findings show removal of hyaluronan in the tumor microenvironment improves immune cells and checkpoint inhibitors access to tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4149/F1.large.jpg.

Nephronectin Regulates Mesangial Cell Adhesion and Behavior in Glomeruli.

A critical aspect of kidney function occurs at the glomerulus, the capillary network that filters the blood. The glomerular basement membrane (GBM) is a key component of filtration, yet our understanding of GBM interactions with mesangial cells, specialized pericytes that provide structural stability to glomeruli, is limited. We investigated the role of nephronectin (Npnt), a GBM component and known ligand of α8ß1 integrin. Immunolocalization and in situ hybridization studies in kidneys of adult mice revealed that nephronectin is produced by podocytes and deposited into the GBM. Conditional deletion of Npnt from nephron progenitors caused a pronounced increase in mesangial cell number and mesangial sclerosis. Nephronectin colocalized with α8ß1 integrin to novel, specialized adhesion structures that occurred at sites of mesangial cell protrusion at the base of the capillary loops. Absence of nephronectin disrupted these adhesion structures, leading to mislocalization of α8ß1. Podocyte-specific deletion of Npnt also led to mesangial sclerosis in mice. These results demonstrate a novel role for nephronectin and α8ß1 integrin in a newly described adhesion complex and begin to uncover the molecular interactions between the GBM and mesangial cells, which govern mesangial cell behavior and may have a role in pathologic states.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.

Role of CD34 family members in lumen formation in the developing kidney.

Previous studies have shown CD34 family member Podocalyxin is required for epithelial lumen formation in vitro. We demonstrate that Endoglycan, a CD34 family member with homology to Podocalyxin, is produced prior to lumen formation in developing nephrons. Endoglycan localizes to Rab11-containing vesicles in nephron progenitors, and then relocalizes to the apical surface as progenitors epithelialize. Once an apical/luminal surface is formed, Endoglycan (and the actin-binding protein Ezrin) localize to large, intraluminal structures that may be vesicles/exosomes. We generated mice lacking Endoglycan and found mutants had timely initiation of lumen formation and continuous lumens, similar to controls. Mice with conditional deletion of both Endoglycan and Podocalyxin in developing nephrons also had normal tubular lumens. Despite this, Endoglycan/Podocalyxin is required for apical recruitment of the adaptor protein NHERF1, but not Ezrin, in podocyte precursors, a subset of the epithelia. In summary, while CD34 family members appear dispensable for lumen formation, our data identify Endoglycan as a novel pre-luminal marker and suggest lumen formation occurs via vesicular trafficking of apical cargo that includes Endoglycan.

Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production.

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε (DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531-536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377-384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.

Accumulation of extracellular hyaluronan by hyaluronan synthase 3 promotes tumor growth and modulates the pancreatic cancer microenvironment.

Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic ß-catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and ß-catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth.

De novo lumen formation and elongation in the developing nephron: a central role for afadin in apical polarity.

A fundamental process in biology is the de novo formation and morphogenesis of polarized tubules. Although these processes are essential for the formation of multiple metazoan organ systems, little is known about the molecular mechanisms that regulate them. In this study, we have characterized several steps in tubule formation and morphogenesis using the mouse kidney as a model system. We report that kidney mesenchymal cells contain discrete Par3-expressing membrane microdomains that become restricted to an apical domain, coinciding with lumen formation. Once lumen formation has been initiated, elongation occurs by simultaneous extension and additional de novo lumen generation. We demonstrate that lumen formation and elongation require afadin, a nectin adaptor protein implicated in adherens junction formation. Mice that lack afadin in nephron precursors show evidence of Par3-expressing membrane microdomains, but fail to develop normal apical-basal polarity and generate a continuous lumen. Absence of afadin led to delayed and diminished integration of nectin complexes and failure to recruit R-cadherin. Furthermore, we demonstrate that afadin is required for Par complex formation. Together, these results suggest that afadin acts upstream of the Par complex to regulate the integration and/or coalescence of membrane microdomains, thereby establishing apical-basal polarity and lumen formation/elongation during kidney tubulogenesis.

Stem cell research ethics: consensus statement on emerging issues.

This article is a consensus statement by an international interdisciplinary group of academic experts and Canadian policy-makers on emerging ethical, legal and social issues in human embryonic stem cells (hESC) research in Canada. The process of researching consensus included consultations with key stakeholders in hESC research (regulations, stem cell researchers, and research ethics experts), preparation and distribution of background papers, and an international workshop held in Montreal in February 2007 to discuss the papers and debate recommendations. The recommendations provided in the consensus statement focus on issues of immediate relevance to Canadian policy-makers, including informed consent to hESC research, the use of fresh embryos in research, management of conflicts of interest, and the relevance of public opinion research to policy-making.