HIF-1 inactivation empowers HIF-2 to drive hypoxia adaptation in aggressive forms of medulloblastoma.

Medulloblastoma (MB) is the most prevalent brain cancer in children. Four subgroups of MB have been identified; of these, Group 3 is the most metastatic. Its genetics and biology remain less clear than the other groups, and it has a poor prognosis and few effective treatments available. Tumor hypoxia and the resulting metabolism are known to be important in the growth and survival of tumors but, to date, have been only minimally explored in MB. Here we show that Group 3 MB tumors do not depend on the canonical transcription factor hypoxia-inducible factor-1α (HIF-1α) to mount an adaptive response to hypoxia. We discovered that HIF-1α is rendered inactive either through post-translational methylation, preventing its nuclear localization specifically in Group 3 MB, or by a low expression that prevents modulation of HIF-target genes. Strikingly, we found that HIF-2 takes over the role of HIF-1 in the nucleus and promotes the activation of hypoxia-dependent anabolic pathways. The exclusion of HIF-1 from the nucleus in Group 3 MB cells enhances the reliance on HIF-2's transcriptional role, making it a viable target for potential anticancer strategies. By combining pharmacological inhibition of HIF-2α with the use of metformin, a mitochondrial complex I inhibitor to block respiration, we effectively induced Group 3 MB cell death, surpassing the effectiveness observed in Non-Group 3 MB cells. Overall, the unique dependence of MB cells, but not normal cells, on HIF-2-mediated anabolic metabolism presents an appealing therapeutic opportunity for treating Group 3 MB patients with minimal toxicity.

The mitochondrial NADH shuttle system is a targetable vulnerability for Group 3 medulloblastoma in a hypoxic microenvironment.

Medulloblastoma is a cancerous brain tumor that affects mostly children. Among the four groups defined by molecular characteristics, Group 3, the least well characterized, is also the least favorable, with a survival rate of 50%. Current treatments, based on surgery, radiotherapy, and chemotherapy, are not adequate and the lack of understanding of the different molecular features of Group 3 tumor cells makes the development of effective therapies challenging. In this study, the problem of medulloblastoma is approached from a metabolic standpoint in a low oxygen microenvironment. We establish that Group 3 cells use both the mitochondrial glycerol-3 phosphate (G3PS) and malate-aspartate shuttles (MAS) to produce NADH. Small molecules that target G3PS and MAS show a greater ability to decrease cell proliferation and induce apoptosis specifically of Group 3 cells. In addition, as Group 3 cells show improved respiration in hypoxia, the use of Phenformin, a mitochondrial complex 1 inhibitor, alone or in combination, induced significant cell death. Furthermore, inhibition of the cytosolic NAD+ recycling enzyme lactate dehydrogenase A (LDHA), enhanced the effects of the NADH shuttle inhibitors. In a 3D model using Group 3 human cerebellar organoids, tumor cells also underwent apoptosis upon treatment with NADH shuttle inhibitors. Our study demonstrates metabolic heterogeneity depending on oxygen concentrations and provides potential therapeutic solutions for patients in Group 3 whose tumors are the most aggressive.

HIF-1 inactivation empowers HIF-2 to drive hypoxia adaptation in aggressive forms of medulloblastoma.

Medulloblastoma (MB) is the most prevalent brain cancer in children. Four subgroups of MB have been identified; of these, Group 3 is the most metastatic. Its genetics and biology remain less clear than the other groups, and it has a poor prognosis and few effective treatments available. Tumor hypoxia and the resulting metabolism are known to be important in the growth and survival of tumors but, to date, have been only minimally explored in MB. Here we show that Group 3 MB tumors do not depend on the canonical transcription factor hypoxia-inducible factor-1α (HIF-1α) to mount an adaptive response to hypoxia. We discovered that HIF-1α is rendered inactive either through post-translational methylation, preventing its nuclear localization specifically in Group 3 MB, or by a low expression that prevents modulation of HIF-target genes. Strikingly, we found that HIF-2 takes over the role of HIF-1 in the nucleus and promotes the activation of hypoxia-dependent anabolic pathways. The exclusion of HIF-1 from the nucleus in Group 3 MB cells enhances the reliance on HIF-2's transcriptional role, making it a viable target for potential anticancer strategies. By combining pharmacological inhibition of HIF-2α with the use of metformin, a mitochondrial complex I inhibitor to block respiration, we effectively induced Group 3 MB cell death, surpassing the effectiveness observed in Non-Group 3 MB cells. Overall, the unique dependence of MB cells, but not normal cells, on HIF-2-mediated anabolic metabolism presents an appealing therapeutic opportunity for treating Group 3 MB patients with minimal toxicity.

MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways.

Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the overexpression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of ß-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-ß-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two 'oncomirs' may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer.

To define novel pathways that regulate susceptibility to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in non-small cell lung cancer (NSCLC), we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant NSCLC cells, levels of different miRs are increased, and in particular, miR-221 and -222. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. Indeed, transfection with anti-miR-221 and -222 rendered CALU-1-resistant cells sensitive to TRAIL. Conversely, H460-sensitive cells treated with -221 and -222 pre-miRs become resistant to TRAIL. miR-221 and -222 target the 3'-UTR of Kit and p27(kip1) mRNAs, but interfere with TRAIL signaling mainly through p27(kip1). In conclusion, we show that high expression levels of miR-221 and -222 are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets or diagnostic tool for TRAIL resistance in NSCLC.

[Mortality in a cohort of asbestos cement workers in Carrara, Italy]. / Studio sulla mortalità degli addetti alla produzione in un'azienda di manufatti in cemento amianto a Carrara.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the mortality of 262 workers (200 men and 62 women) employed in an asbestos cement plant located in Carrara, Italy, exposed to a mixture of chrysotile and crocidolite asbestos in a ratio of 2:5. METHODS: Follow-up started on 1 January, 1963. The vital status and causes of death were ascertained on 31 December, 2003. The Tuscany population mortality was used as reference. The relative risk was estimated by Standardized Mortality Ratio (SMR) and the confidence intervals were calculated at 95% level [95%CI]. RESULTS: Among men, a significant increase in mortality was observed for respiratory disease (14 deaths; SMR = 244.1; IC95% = 133.4-409.5), particularly for pneumoconiosis (10 deaths; SMR= 1,800; IC95% = 856.9-3,300.0; of which 5 deaths due to asbestosis; SMR = 120,000; IC95% = 37,000-270,000), and for pleural cancer (4 deaths; SMR = 2,500; IC95% = 676.8-6,400.0). Non-significant increases were also observed for lung cancer (10 deaths; SMR = 114.2; IC95% = 54.8-209.9), and gastric cancer (7 deaths; SMR= 167.1; IC95% = 67.2-344.3). Among women, significant increases were observed for pneumoconiosis (1 death; SMR = 17,000; 95%CI = 425-93,000), and for liver cancer (3 deaths; SMR = 765.0; IC95% = 157.8-2,200.0). CONCLUSIONS: For males our results were consistent with other mortality studies on asbestos-cement workers. No other cohort studies on asbestos cement workers have dealt with mortality of female workers.

Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase alpha (PI3Kalpha ). Functions of lipid kinase-deficient PI3Kalpha in signaling.

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.

Analysis of the murine phosphoinositide 3-kinase gamma gene.

Phosphoinositide 3-kinase gamma is preferentially expressed in leukocytes. PI3Kgamma is activated by betagamma subunits of heterotrimeric G-proteins, which thus link seven transmembrane helix receptor activation to phosphatidylinositol (3,4,5)-trisphosphate production. Here we describe the molecular cloning of the murine PI3Kgamma cDNA, the PI3Kgamma gene structure, its chromosomal assignment and the analysis of promoter activity. The mouse cDNA shares 86% identity to its pig and human orthologues at the nucleotide level. The MmPI3Kgamma gene spans approximately 30kb and comprises 11 exons. RACE-PCR indicated the presence of multiple start sites generating 5' UTRs with different lengths, the longest being 874bp. The putative promoter region contains no TATA box but several putative binding sites for hematopoietic specific transcription factors. A 1200bp long sequence upstream the first transcription start site was found to possess tissue specific promoter activity. Deletion constructs revealed two contiguous regions, with activator function, ranging from positions -139 to -557, and with inhibitory function, ranging from positions -557 to -892. FISH analysis revealed that the MmPI3Kgamma is located on chromosome 12 band B and that the human orthologue is positioned on chromosome 7q22.2-22.3. In spite of some differences in the ATP-binding site, recombinant murine PI3Kgamma protein is equally sensitive to wortmannin as its human counterpart. This suggests that mouse models will provide reliable results in the assessments of novel PI3Kgamma inhibitors.

[Ultrasonographic study of detrusor thickness in patients with obstruction]. / Studio ecografico dello spessore del detrusore nel paziente ostruito.

The diagnostic work up of patients with symptomatic benign prostatic hyperplasia is not always useful for diagnosing bladder outlet obstruction, especially if they are compared with pressure-flow studies. The authors make a review of the literature and a clinical study to demonstrate that alternative less invasive methods (ultrasound estimated bladder weight) can often replace pressure/flow studies.

Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK.

Phosphoinositide 3-kinases (PI3Ks) activate protein kinase PKB (also termed Akt), and PI3Kgamma activated by heterotrimeric guanosine triphosphate-binding protein can stimulate mitogen-activated protein kinase (MAPK). Exchange of a putative lipid substrate-binding site generated PI3Kgamma proteins with altered or aborted lipid but retained protein kinase activity. Transiently expressed, PI3Kgamma hybrids exhibited wortmannin-sensitive activation of MAPK, whereas a catalytically inactive PI3Kgamma did not. Membrane-targeted PI3Kgamma constitutively produced phosphatidylinositol 3,4, 3,4,5-trisphosphate and activated PKB but not MAPK. Moreover, stimulation of MAPK in response to lysophosphatidic acid was blocked by catalytically inactive PI3Kgamma but not by hybrid PI3Kgammas. Thus, two major signals emerge from PI3Kgamma: phosphoinositides that target PKB and protein phosphorylation that activates MAPK.

Lipid kinase and protein kinase activities of G-protein-coupled phosphoinositide 3-kinase gamma: structure-activity analysis and interactions with wortmannin.

Signalling via seven transmembrane helix receptors can lead to a massive increase in cellular PtdIns(3,4,5)P3, which is critical for the induction of various cell responses and is likely to be produced by a trimeric G-protein-sensitive phosphoinositide 3-kinase (PI3Kgamma). We show here that PI3Kgamma is a bifunctional lipid kinase and protein kinase, and that both activities are inhibited by wortmannin at concentrations equal to those affecting the p85/p110alpha heterodimeric PI3K (IC50 approx. 2 nM). The binding of wortmannin to PI3Kgamma, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. Truncation of more than the 98 N-terminal amino acid residues from PI3Kgamma produced proteins that were inactive in wortmannin binding and kinase assays. This suggests that regions apart from the core catalytic domain are important in catalysis and inhibitor interaction. The covalent reaction of wortmannin with PI3Kgamma was prevented by preincubation with phosphoinositides, ATP and its analogues adenine and 5'-(4-fluorosulphonylbenzoyl)adenine. Proteolytic analysis of wortmannin-prelabelled PI3Kgamma revealed candidate wortmannin-binding peptides around Lys-799. Replacement of Lys-799 by Arg through site-directed mutagenesis aborted the covalent reaction with wortmannin and the lipid kinase and protein kinase activities completely. The above illustrates that Lys-799 is crucial to the phosphate transfer reaction and wortmannin reactivity. Parallel inhibition of the PI3Kgamma-associated protein kinase and lipid kinase by wortmannin and by the Lys-799-->Arg mutation reveals that both activities are inherent in the PI3Kgamma polypeptide.

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

[Radical prostatectomy: role of ultrasonography in the follow-up]. / Prostatovesciculectomia radicale: ruolo dell'ecografia nel follow-up.

After a brief summary about surgical technique of radical prostatectomy and its indications in the different stages of prostatic cancer, the Authors describe complications and surgical sequelae of this operation. The Authors report a brief series. It is composed with 13 patients. Their have been submitted to radical prostatectomy for prostatic cancer between January 1989 and September 1991. Pathological stage was B1 in 6 patients, B2 in 2 patients, C1 in 4 cases and C2 in 1. Particularly the role of ultrasonography in detection and follow-up of early complications such as lymphocele, pelvic hematoma and anastomotic urine leakage is stressed. Transrectal ultrasound is especially useful in the detection of urine leakage from vesico-urethral anastomosis. This technique is compared with traditional cystourethrography and advantages and disadvantages of the two techniques are discussed. Later complications of radical prostatectomy are anastomotic stenosis, pelvic recurrences, nodal or parenchymal metastasis, urinary incontinence. The role of transrectal ultrasound in the detection of anastomotic strictures is stressed, especially when the study is done during micturition. Transrectal ultrasound is not so satisfying in the detection of pelvic recurrences, especially if they are smaller than 1 cm. In case of large masses digital examination is diagnostic itself. At last the Authors describe urinary incontinence and its etiology as a complication of radical prostatectomy. Particularly a surgical technique for vesico-urethral anastomosis proposed by Rocca Rossetti and its value in post-operative continence is described. The Authors show the results of transrectal ultrasound in the detection of striated urethral sphincter and its function after radical prostatectomy.

[Mono-symptomatic hematuria: first symptom of metastatic pulmonary carcinoma]. / Ematuria monosintomatica: primo sintomo di carcinoma polmonare metastatico.

The occurrence of secondary bladder neoplasms is very uncommon, especially when the bladder is the only site of metastasis. The Authors report on one case of bladder metastasis from primary small cell carcinoma of the lung.

[Cystic nephroma: report of two cases and review of literature]. / Nefroma cistico: nostra esperienza su due casi e revisione della letteratura.

Cystic nephroma is an uncommon lesion, whose etiology and pathogenesis is still debated: some Authors designate it as being of neoplastic origin, other ones of dysplastic or hamartomous origin. Also epidemiology makes difficult its pathogenetic interpretation, as being especially affected children under age of fourth year and adults within the 5th and 6th decade. The Authors report two cases of cystic nephroma examined in two female patients 30 and 74 aged. The most interesting matters are: 1) Possibility of a pre-operative diagnosis of founded suspicion, based on pathologic criteria, codified in literature (unilateral and multilocular cyst which doesn't communicate with the renal collecting system, separated by delicate septae without mature renal tissue) and on respective ultrasonographic, CT and angiographic patterns; 2) Possibility of programming a surgical-conservative strategy; 3) Knowledge about possibility of foci association of adeno-carcinoma or nephroblastoma in the lesion, that, nevertheless, if not widespread, it should not modified neither therapeutical proceeding nor prognosis, generally favourable.

[Extrinsic ureteral endometriosis: description of a case]. / Endometriosi ureterale estrinseca: descrizione di un caso.

After a brief review about pathogenetic hypothesis of the endometriosis of the ureter, the Authors describe a case occurred to their observation. Diagnostic problems and choice in treatment (especially partial ureterectomy, end-to-end ureteral anastomosis and omentoplasty) are discussed.

[Experience with the treatment of infertility]. / Opyt lecheniia bo'lnykh s besplodiem.

Pathogenetic treatment policy was determined on the basis of the research data obtained from 1210 patients exposed to laparoscopy in combination with chromosalpingoscopy and biopsy of the ovary. The combined treatment resulted in the recovery of reproductive function in 52 per cent of the patients.