Michelangelo Fiorentino

Androgen Receptor Regulates a Distinct Transcription Program in Androgen-Independent Prostate Cancer

The evolution of prostate cancer from an androgen-dependent state to one that is androgen-independent marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in androgen-independent cancers is poorly understood. We have defined the direct AR-dependent target genes in both androgen-dependent and -independent cancer cells by generating AR-dependent gene expression profiles and AR cistromes. In contrast to what is found in androgen-dependent cells, AR selectively upregulates M-phase cell-cycle genes in androgen-independent cells, including UBE2C, a gene that inactivates the M-phase checkpoint. We find that epigenetic marks at the UBE2C enhancer, notably histone H3K4 methylation and FoxA1 transcription factor binding, are present in androgen-independent cells and direct AR-enhancer binding and UBE2C activation. Thus, the role of AR in androgen-independent cancer cells is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

Background Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. Methodology We developed and implemented an optimized mutation profiling platform (“OncoMap”) to interrogate ∼400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. Conclusions Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of “actionable” cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.

Met Activation in Non-Small Cell Lung Cancer Is Associated with de Novo Resistance to EGFR Inhibitors and the Development of Brain Metastasis

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.

Partial necrosis on hepatocellular carcinoma nodules facilitates tumor recurrence after liver transplantation.

Background. The presence of partial necrosis in hepatocellular carcinoma (HCC) nodules is a common histologic finding after liver transplantation, but its correlation with tumor recurrence has never been investigated. Methods. we retrospectively reviewed the outcome of 54 patients with a single histologically proven HCC after liver transplantation. All cases had a survival of more than 6 months, and patients treated preoperatively had a transarterial chemoembolization (TACE) procedure. Since 1996, our center has applied the Milan criteria. Correlations between tumor recurrences and clinicopathologic variables, including the presence of partial necrosis, were performed. Etiologic factors for HCC partial necrosis were also investigated. Results. Sixteen of 54 (29.6%) HCC nodules presented partial necrosis, and 4 (25%) of them developed HCC recurrence compared with 1 of 38 (2.6%) cases without this histologic finding (P<0.05). Partial necrosis was related to TACE procedure (P<0.05), patient age less than 50 years (P<0.05), and tumor diameter greater than 2 cm (P<0.05). Multivariate analysis showed only TACE as an independent variable. The other variables related to the five (9.3%) tumor recurrences were HCC diameter greater than 2 cm (P<0.05), year of liver transplantation before 1996 (P<0.05), and the presence of satellite nodules (P<0.05). The Cox regression analysis showed the presence of partial necrosis as an independent variable related to tumor recurrence. The analysis of the recurrence-free survival confirmed the results of the recurrence rate. Conclusion. Partial necrosis was a risk factor for tumor recurrence after liver transplantation. Patients and procedures should be selected while also bearing in mind the side-effect of incomplete necrosis of the nodules.

Fatty Acid Synthase Polymorphisms, Tumor Expression, Body Mass Index, Prostate Cancer Risk, and Survival

PURPOSE Fatty acid synthase (FASN) regulates de novo lipogenesis, body weight, and tumor growth. We examined whether common germline single nucleotide polymorphisms (SNPs) in the FASN gene affect prostate cancer (PCa) risk or PCa-specific mortality and whether these effects vary by body mass index (BMI). METHODS In a prospective nested case-control study of 1,331 white patients with PCa and 1,267 age-matched controls, we examined associations of five common SNPs within FASN (and 5 kb upstream/downstream, R(2) > 0.8) with PCa incidence and, among patients, PCa-specific death and tested for an interaction with BMI. Survival analyses were repeated for tumor FASN expression (n = 909). RESULTS Four of the five SNPs were associated with lethal PCa. SNP rs1127678 was significantly related to higher BMI and interacted with BMI for both PCa risk (P(interaction) = .004) and PCa mortality (P(interaction) = .056). Among overweight men (BMI > or = 25 kg/m(2)), but not leaner men, the homozygous variant allele carried a relative risk of advanced PCa of 2.49 (95% CI, 1.00 to 6.23) compared with lean men with the wild type. Overweight patients carrying the variant allele had a 2.04 (95% CI, 1.31 to 3.17) times higher risk of PCa mortality. Similarly, overweight patients with elevated tumor FASN expression had a 2.73 (95% CI, 1.05 to 7.08) times higher risk of lethal PCa (P(interaction) = .02). CONCLUSION FASN germline polymorphisms were significantly associated with risk of lethal PCa. Significant interactions of BMI with FASN polymorphisms and FASN tumor expression suggest FASN as a potential link between obesity and poor PCa outcome and raise the possibility that FASN inhibition could reduce PCa-specific mortality, particularly in overweight men.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of β-catenin in prostate cancer

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that 14C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic β-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of β-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed β-catenin. A strong significant association between FASN and cytoplasmic (stabilized) β-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous β-catenin signal (P<0.001, Spearmans ρ=0.33). We propose that cytoplasmic stabilization of β-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.

Probiotic therapy in the prevention of pouchitis onset : Decreased interleukin-1β, interleukin-8, and interferon-γ gene expression

Background: Probiotic therapy has been shown to prevent the onset of pouchitis and to improve the quality of life in ulcerative colitis patients who required ileal pouch anal anastomosis. Pouchitis has been associated with elevated levels of proinflammatory cytokines and chemokines. Methods: In this retrospective analysis of archived endoscopic samples from responding patients enrolled in the above‐mentioned trial, we were interested in studying mucosal gene expression of the pleiotropic proinflammatory cytokines (interleukin‐1&bgr;, interleukin‐6), TH1 cytokines (interferon‐&ggr;, tumor necrosis factor‐&agr;, interleukin‐12), regulatory cytokines (interleukin‐10, transforming growth factor‐&bgr;), and the chemokine interleukin‐8. In addition to assessment of cytokine gene expression, the presence of polymorphonuclear cells in the mucosal tissue was evaluated. Results: Data show that patients who were treated with probiotics had significant lower mucosal mRNA expression levels of interleukin‐1&bgr;, interleukin‐8, and interferon‐&ggr; compared with placebo‐treated patients. Conclusions: In addition, a lower number of polymorphonuclear cells was present in the tissue of patients within the probiotic group compared with the number of polymorphonuclear cells in the tissue of patients receiving placebo and patients having an episode of pouchitis. Conclusions: These data suggest that probiotic treatment regulates the mucosal immune response by reducing mucosal levels of neutrophil‐chemoattractant IL‐8 and tissue influx of polymorphonuclear cells, and may further act by inhibition of T‐cell activation, by reinforcement of barrier function and by a tight control of the potent pro‐inflammatory cytokine IL‐1&bgr;.

Diagnostic role of circulating free plasma DNA detection in patients with localized prostate cancer

To analyze the potential diagnostic relevance of free plasma DNA (FPDNA), we enrolled 64 patients with localized prostate cancer (CaP). FPDNA was quantified by real-time polymerase chain reaction assessment of the HTERT gene in blood samples from 64 patients with CaP and 45 healthy males. Methylation of the GSTP1 gene was used to confirm the neoplastic origin of FPDNA in selected cases. The mean +/- SD levels of FPDNA were higher in patients with CaP (15.4 +/- 10.9 ng/mL) than in control subjects (5.5 +/- 3.5 ng/mL; P <.001). By using the best cutoff value, the sensitivity of the test was 80%, the specificity was 82%, the area under the receiver operating characteristic curve, 0.881. High FPDNA values were significantly associated with pathologic T3 stage (P = . 035). Methylation of the GSTP1 gene was found in 4 (25%) of 16 FPDNA samples and 15 (94%) of 16 tissue samples. Quantification of FPDNA discriminates between patients with CaP and healthy subjects and correlates with pathologic tumor stage. FPDNA is a candidate biomarker for early diagnosis and monitoring of CaP.

Loss of miR-101 expression promotes Wnt/β-catenin signalling pathway activation and malignancy in colon cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer‐related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR‐101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR‐101 re‐expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR‐101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR‐101 expression and important CRC pro‐malignant features, such as inflammation, activation of the Wnt/β‐catenin signalling pathway and epithelial–mesenchymal transition (EMT). We then propose that up‐regulated miR‐101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR‐101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis. Copyright

Differential requirement of mTOR in postmitotic tissues and tumorigenesis.

Conditional inactivation of mTor has little effect in adult mouse prostate, but suppresses tumor initiation and progression. Blocking mTor Signals The mammalian target of rapamycin (mTOR), a protein kinase critical to cell growth and proliferation, functions as part of two distinct multiprotein complexes. mTOR signaling is frequently disrupted in cancer; however pharmacological suppression of mTOR complex 1 (mTORC1) signaling has been of limited therapeutic efficacy. Nardella et al. show that, whereas conditional inactivation of mTOR activity (abrogating signaling through both complexes) has little effect in the adult mouse prostate, it markedly suppresses prostate cancer associated with loss of the tumor suppressor PTEN. Thus, mTOR inhibitors that target its catalytic activity may be more effective in cancer therapy than those that specifically inhibit signaling mediated through mTORC1. The mammalian target of rapamycin (mTOR) is a crucial effector in a complex signaling network commonly disrupted in cancer. mTOR exerts its multiple functions in the context of two different multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Loss of the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) can hyperactivate mTOR through AKT and represents one of the most frequent events in human prostate cancer. We show here that conditional inactivation of mTor in the adult mouse prostate is seemingly inconsequential for this postmitotic tissue. Conversely, inactivation of mTor leads to a marked suppression of Pten loss–induced tumor initiation and progression in the prostate. This suppression is more pronounced than that elicited by the sole pharmacological abrogation of mTORC1. Acute inactivation of mTor in vitro also highlights the differential requirement of mTor function in proliferating and transformed cells. Collectively, our data constitute a strong rationale for developing specific mTOR inhibitors targeting both mTORC1 and mTORC2 for the treatment of tumors triggered by PTEN deficiency and aberrant mTOR signaling.