Quantifying geographical accessibility to cancer clinical trials in different income landscapes.

BACKGROUND: Clinical trials are increasingly perceived as a therapeutic opportunity for cancer patients. Favoring their concentration in few high-expertise academic centers maximizes quality of data collection but poses an issue of access equality. Analytical tools to quantify trial accessibility are needed to rationalize resources. MATERIALS AND METHODS: We constructed a distance-based accessibility index (dAI) using publicly available data on demographics, cancer incidence and trials. Multiple strategies were applied to mitigate or quantify clear sources of bias: reporting biases by text mining multiple registries; reliability of simple geographical distance by comparison with high-quality travel cost data for Italy; index inflation due to highly heterogeneous cancer incidence by log-transformation. We studied inequalities by Gini index and time trend significance by Mann-Kendall test. We simulated different resource allocation models in representative countries and identified locations where new studies would maximally improve the national index. RESULTS: The dAI approximated well a more realistic but not widely applicable travel cost-based index. Accessibility was unevenly distributed across and within countries (Gini index ∼0.75), with maximal inequalities in high- and upper-middle-income countries (China, United States, Russian Federation). Over time, accessibility increased but less than the total number of trials, most evidently in upper-middle-income countries. Simulations in representative countries (Italy and Serbia) identified ideal locations able to maximally raise the national index. CONCLUSIONS: Access to clinical trials is highly uneven across and within countries and is not mitigated by simple increase in the number of trials; a rational algorithmic approach can be used to mitigate inequalities.

Aging phenotype(s) in kidneys of diabetic mice are p66ShcA dependent.

The p66ShcA protein controls cellular responses to oxidative stress, senescence, and apoptosis. Here, we test the hypothesis that aging phenotype(s) commonly associated with the broad category of chronic kidney disease are accelerated in diabetic kidneys and linked to the p66ShcA locus. At the organ level, tissue stem cells antagonize senescent phenotypes by replacing old dysfunctional cells. Using established methods, we isolated a highly purified population of stem cell antigen-1-positive mesenchymal stem cells (Sca-1+ MSCs) from kidneys of wild-type (WT) and p66 knockout (p66 KO) mice. Cells were plated in culture medium containing normal glucose (NG) or high glucose (HG). Reactive oxygen species (ROS) metabolism was substantially increased in WT MSCs in HG medium in association with increased cell death by apoptosis and acquisition of the senescent phenotype. DNA microarray analysis detected striking differences in the expression profiles of WT and p66 KO-MSCs in HG medium. Unexpectedly, the analysis for p66 KO-MSCs revealed upregulation of Wnt genes implicated in self-renewal and differentiation. To test the in vivo consequences of constitutive p66 expression in diabetic kidneys, we crossed the Akita diabetic mouse with the p66KO mouse. Homozygous mutation at the p66 locus delays or prevents aging phenotype(s) in the kidney that may be precursors to diabetic nephropathy.

The E3 ubiquitin ligase WWP1 sustains the growth of acute myeloid leukaemia.

The E3 ubiquitin ligase (E3) WWP1 is an oncogenic factor implicated in the maintenance of different types of epithelial cancers. The role of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in haematological neoplasms remains unknown. Acute myeloid leukaemia (AML) is characterized by the expansion of malignant myeloid cells blocked at different stages of differentiation. Here we report that the expression of WWP1 is significantly augmented in a large cohort of primary AML patients and in AML cell lines, compared with haematopoietic cells from healthy donors. We show that WWP1 inactivation severely impairs the growth of primary AML blasts and cell lines in vitro. In vivo, we observed a reduced leukaemogenic potential of WWP1-depleted AML cells upon transplantation into immunocompromised mice. Mechanistically, WWP1 inactivation induces the accumulation of its protein substrate p27Kip1, which ultimately contributes to G0/G1 cell cycle arrest of AML blasts. In addition, WWP1 depletion triggers the autophagy signalling and reduces survival of leukaemic cells. Collectively, our findings provide molecular insights into the anti-cancer potential of WWP1 inhibition, suggesting that this E3 is a promising biomarker and druggable target in AML.

PML-RARA-associated cooperating mutations belong to a transcriptional network that is deregulated in myeloid leukemias.

It has been shown that individual acute myeloid leukemia (AML) patients are characterized by one of few initiating DNA mutations and 5-10 cooperating mutations not yet defined among hundreds identified by massive sequencing of AML genomes. We report an in vivo insertional-mutagenesis screen for genes cooperating with one AML initiating mutations (PML-RARA, oncogene of acute promyelocytic leukemia, APL), which allowed identification of hundreds of genetic cooperators. The cooperators are mutated at low frequency in APL or AML patients but are always abnormally expressed in a cohort of 182 APLs and AMLs analyzed. These deregulations appear non-randomly distributed and present in all samples, regardless of their associated genomic mutations. Reverse-engineering approaches showed that these cooperators belong to a single transcriptional gene network, enriched in genes mutated in AMLs, where perturbation of single genes modifies expression of others. Their gene-ontology analysis showed enrichment of genes directly involved in cell proliferation control. Therefore, the pool of PML-RARA cooperating mutations appears large and heterogeneous, but functionally equivalent and deregulated in the majority of APLs and AMLs. Our data suggest that the high heterogeneity of DNA mutations in APLs and AMLs can be reduced to patterns of gene expression deregulation of a single 'mutated' gene network.

Cyclophilin D counteracts P53-mediated growth arrest and promotes Ras tumorigenesis.

Mitochondrial alterations induced by oncogenes are known to be crucial for tumorigenesis. Ras oncogene leads to proliferative signals through a Raf-1/MEK/ERK kinase cascade, whose components have been found to be also associated with mitochondria. The mitochondrial pepdidyl-prolyl isomerase cyclophilin D (CypD) is an important regulator of the mitochondrial permeability transition and a key player in mitochondria physiology; however, its role in cancer is still unclear. Using cellular and in vivo mouse models, we demonstrated that CypD protein upregulation induced by oncogenic Ras through the Raf-1/MEK/ERK pathway has a deterministic role in tumor progression. In fact, targeting CypD gene expression clearly affected RasV12-induced transformation, as showed by in vitro data on murine NIH3T3 and human MCF10A mammary epithelial cells. In addition, studies in xenograft and K-Ras lung cancer mouse models demonstrated that genetic deletion or pharmacological suppression of CypD efficiently prevented Ras-dependent tumor formation. Furthermore, Erbb2-mediated breast tumorigenesis was similarly prevented by targeting CypD. From a mechanistic point of view, CypD expression was associated with a reduced induction of p21(WAF1/CIP1) and p53 functions, unraveling an antagonistic function of CypD on p21-p53-mediated growth suppression. CypD activity is p53 dependent. Interestingly, a physical association between p53 and CypD was detected in mitochondria of MCF10A cells; furthermore, both in vitro and in vivo studies proved that CypD inhibitor-based treatment was able to efficiently impair this interaction, leading to a tumor formation reduction. All together, these findings indicate that the countering effect of CypD on the p53-p21 pathway participates in oncogene-dependent transformation.

PML is required for telomere stability in non-neoplastic human cells.

Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.

Negative regulation of chemokine receptor signaling and B-cell chemotaxis by p66Shc.

Shc (Src homology 2 domain containing) adaptors are ubiquitous components of the signaling pathways triggered by tyrosine kinase-coupled receptors. In lymphocytes, similar to other cell types, the p52 and p66 isoforms of ShcA/Shc participate in a self-limiting loop where p52Shc acts as a positive regulator of antigen receptor signaling by promoting Ras activation, whereas p66Shc limits this activity by competitively inhibiting p52Shc. Based on the fact that many signaling mediators are shared by antigen and chemokine receptors, including p52Shc, we have assessed the potential implication of p66Shc in the regulation of B-cell responses to chemokines, focusing on the homing receptors CXCR4 (C-X-C chemokine receptor type 4) and CXCR5 (C-X-C chemokine receptor type 5). The results identify p66Shc as a negative regulator of the chemotactic responses triggered by these receptors, including adhesion, polarization and migration. We also provide evidence that this function is dependent on the ability of p66Shc to interact with the chemokine receptors and promote the assembly of an inhibitory complex, which includes the phosphatases SHP-1 (Src homology phosphatase-1) and SHIP-1 (SH2 domain-containing inositol 5'-phosphatase-1), that results in impaired Vav-dependent reorganization of the actin cytoskeleton. This function maps to the phosphorylatable tyrosine residues in the collagen homology 1 (CH1) domain. The results identify p66Shc as a negative regulator of B-cell chemotaxis and suggest a role for this adaptor in the control of B-cell homing.

Gene expression profiling reveals GC and CEACAM1 as new tools in the diagnosis of lung carcinoids.

BACKGROUND: Classification of lung carcinoids into typical and atypical is a diagnostic challenge since no immunohistochemical tools are available to support pathologists in distinguishing between the two subtypes. A differential diagnosis is essential for clinicians to correctly discuss therapy, prognosis and follow-up with patients. Indeed, the distinction between the two typical and atypical subtypes on biopsies/cytological specimens is still unfeasible and sometimes limited also after radical surgeries. By comparing the gene expression profile of typical (TC) and atypical carcinoids (AC), we intended to find genes specifically expressed in one of the two subtypes that could be used as diagnostic markers. METHODS: Expression profiling, with Affymetrix arrays, was performed on six typical and seven atypical samples. Data were validated on an independent cohort of 29 tumours, by means of quantitative PCR and immunohistochemistry (IHC). RESULTS: High-throughput gene expression profiling was successfully used to identify a gene signature specific for atypical lung carcinoids. Among the 273 upregulated genes in the atypical vs typical subtype, GC (vitamin D-binding protein) and CEACAM1 (carcinoembryonic antigen family member) emerged as potent diagnostic markers. Quantitative PCR and IHC on a validation set of 17 ACs and 12 TCs confirmed their reproducibility and feasibility. CONCLUSIONS: GC and CEACAM1 can distinguish between TC and AC, defining an IHC assay potentially useful for routine cytological and histochemical diagnostic procedures. The high sensitivity and reproducibility of this new diagnostic algorithm strongly support a further validation on a wider sample size.

Hepatocyte odd protein shuttling (HOPS) is a bridging protein in the nucleophosmin-p19 Arf network.

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19(Arf), directs localization of p19(Arf) in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19(Arf), resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19(Arf) and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19(Arf), providing new mechanistic insight into how NPM and p19(Arf) will oppose tumor cell proliferation.

Normal and leukemic stem cells.

Studies on hematopoietic stem cells have provided several critical insights in the biology of stem cells in general; as mature blood cells are generally short lived, stem cells are in fact required to guarantee, throughout the life of an organism, the replenishment of differentiated blood cells by the generation of multi-lineage progenitors and precursors committed to individual hematopoietic lineages. Similarly, acute myeloid leukemia has been considered as a model system to study cancer stem cells. This presentation illustrates some recent results obtained by our group with regard to both normal and leukemic stem cells.

Genomic instability in induced stem cells.

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.

The self-association coiled-coil domain of PML is sufficient for the oncogenic conversion of the retinoic acid receptor (RAR) alpha.

In acute promyelocytic leukemia (APL) the retinoic acid receptor alpha (RARα) becomes an oncogene through the fusion with several partners, mostly with promyelocytic leukemia protein (PML), all of which have in common the presence of a self-association domain. The new fusion proteins, therefore, differently from the wild-type RARα, which forms only heterodimers with retinoic X receptor alpha, are also able to homo-oligomerize. The presence of such a domain has been suggested to be crucial for the leukemogenic potential of the chimeric proteins found in APL blasts. Whether or not any self-association domain is sufficient to bestow a leukemogenic activity on RARα is still under investigation. In this work, we address this question using two different X-RARα chimeras, where X represents the coiled-coil domain of PML (CC-RARα) or the oligomerization portion of the yeast transcription factor GCN4 (GCN4-RARα). We demonstrate that in vitro both proteins have transforming potential, and recapitulate the main PML-RARα biological properties, but CC-RARα is uniquely able to disrupt PML nuclear bodies. Indeed, in vivo only the CC-RARα chimera induces efficiently APL in a murine transplantation model. Thus, the PML CC domain represents the minimal structural determinant indispensable to transform RARα into an oncogenic protein.

Nucleophosmin and its complex network: a possible therapeutic target in hematological diseases.

Nucleophosmin (NPM, also known as B23, numatrin or NO38) is a ubiquitously expressed phosphoprotein belonging to the nucleoplasmin family of chaperones. NPM is mainly localized in the nucleolus where it exerts many of its functions, but a proportion of the protein continuously shuttles between the nucleus and the cytoplasm. A growing number of cellular proteins have been described as physical interactors of NPM, and consequently, NPM is thought to have a relevant role in diverse cellular functions, including ribosome biogenesis, centrosome duplication, DNA repair and response to stress. NPM has been implicated in the pathogenesis of several human malignancies and intriguingly, it has been described both as an activating oncogene and a tumor suppressor, depending on cell type and protein levels. In fact, increased NPM expression is associated with different types of solid tumors whereas an impairment of NPM function is characteristic of a subgroup of hematolologic malignancies. A large body of experimental evidence links the deregulation of specific NPM functions to cellular transformation, yet the molecular mechanisms through which NPM contributes to tumorigenesis remain elusive. In this review, we have summarized current knowledge concerning NPM functions, and attempted to interpret its multifaceted and sometimes apparently contradictory activities in the context of both normal cellular homeostasis and neoplastic transformation.

Genomic characterization of asymptomatic CT-detected lung cancers.

Computed tomography (CT) screening of lung cancer allows the detection of early tumors. The objective of our study was to verify whether initial asymptomatic lung cancers, identified by high-resolution low-dose CT (LD-CT) on a high-risk population, show genetic abnormalities that could be indicative of the early events of lung carcinogenesis. We analyzed 78 tumor samples: 21 (pilot population) from heavy smokers with asymptomatic non-screening detected early-stage lung cancers and 57 from 5203 asymptomatic heavy smoker volunteers, who underwent a LD-CT screening study. During surgical resection of the detected tumors, tissue samples were collected and short-term cultures were started for karyotype evaluation. Samples were classified according to the normal (NK) or aneuploid (AK) karyotype. The NK samples were further analyzed by the Affymetrix single-nucleotide polymorphisms (SNPs) technology. Metaphase spreads were obtained in 73.0% of the selected samples: 80.7% showed an AK. A statistically significant correlation was found between presence of vascular invasion and abnormal karyotype. A total of 10 NK samples were suitable for SNPs analysis. Subtle genomic alterations were found in eight tumors, the remaining two showing no evidence to date of chromosomal aberrations anywhere in the genome. Two common regions of amplification were identified at 5p and 8p11. Mutation analysis by direct sequencing was conducted for the K-RAS, TP53 and EGFR genes, confirming data already described for heavy smokers. We show that: (i) the majority of screening-detected tumors are aneuploid; (ii) early-stage tumors tend to harbor a less abnormal karyotype; (iii) whole genome analysis of NK tumors allows for the detection of common regions of copy number variation (such as amplifications at 5p and 8p11), highlighting genes that might be considered candidate markers of early events in lung carcinogenesis.