Valentina Vaira

Regulation of survivin expression by IGF-1/mTOR signaling

Survivin is a dual regulator of cell proliferation and cell viability overexpressed in most human tumors. Although strategies to lower survivin levels have been pursued for rational cancer therapy, the molecular circuitries controlling survivin expression in tumors have not been completely elucidated. Here, we show that stimulation with insulin-like growth factor-1 (IGF-1) results in increased survivin expression in prostate cancer cells. This response is independent of de novo gene transcription, changes in mRNA expression or modifications of survivin protein stability. Instead, IGF-1 induced persistence and translation of a pool of survivin mRNA, in a reaction abolished by the mTOR (mammalian target of rapamycin) inhibitor, rapamycin. Forced expression of the mTOR target p70S6K1 reproduced the increase in survivin expression in prostate cancer cells, whereas acute ablation of endogenous p70S6K1 by small interfering RNA downregulated survivin levels. Rapamycin, alone or in combination with suboptimal concentrations of taxol reduced survivin protein levels, and decreased viability of prostate cancer cells. Therefore, IGF-1/mTOR signaling elevates survivin in prostate cancer cells via rapid changes in mRNA translation. Antagonists of this pathway may be beneficial to lower an antiapoptotic threshold maintained by survivin in prostate cancer.

Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors

Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial–stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.

Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma

BackgroundSimilarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of hepatocellular carcinoma (HCC). The members of IAP family are key regulators of apoptosis, cytokinesis and signal transduction. IAP survival action is antagonized by specific binding of Smac/DIABLO and XAF1. This study aimed to investigate the gene and protein expression pattern of IAP family members and their antagonists in a series of human HCCs and to assess their clinical significance.MethodsRelative quantification of IAPs and their antagonist genes was assessed by quantitative Real Time RT-PCR (qPCR) in 80 patients who underwent surgical resection for HCC. The expression ratios of XIAP/XAF1 and of XIAP/Smac were also evaluated. Survivin, XIAP and XAF1 protein expression were investigated by immunohistochemistry. Correlations between mRNA levels, protein expression and clinicopathological features were assessed. Follow-up data were available for 69 HCC patients. The overall survival analysis was estimated according to the Kaplan-Meier method.ResultsSurvivin and Livin/ML-IAP mRNAs were significantly over-expressed in cancer tissues compared to non-neoplastic counterparts. Although Survivin immunoreactivity did not correlate with qPCR data, a significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion.The mRNA ratio XIAP/XAF1 was significantly higher in HCCs than in cirrhotic tissues. Moreover, high XIAP/XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival.ConclusionOur study demonstrates that alterations in the expression of IAP family members, including Survivin and Livin/ML-IAP, are frequent in HCCs. Of interest, we could determine that an imbalance in XIAP/XAF1 mRNA expression levels correlated to overall patient survival, and that high XIAP immunoreactivity was a poor prognostic factor.

Pseudohypoparathyroidism and GNAS Epigenetic Defects: Clinical Evaluation of Albright Hereditary Osteodystrophy and Molecular Analysis in 40 Patients

CONTEXT The two main subtypes of pseudohypoparathyroidism (PHP), PHP-Ia and -Ib, are caused by mutations in GNAS exons 1-13 and methylation defects in the imprinted GNAS cluster, respectively. PHP-Ia patients show Albright hereditary osteodystrophy (AHO) and resistance toward PTH and additional hormones, whereas PHP-Ib patients do not have AHO, and hormone resistance appears to be limited to PTH and TSH. Recently, methylation defects have been detected in few patients with PHP and mild AHO, indicating a molecular overlap between the two forms. OBJECTIVES The aim of the study was to screen patients with clinically diagnosed PHP-Ia for methylation defects and to investigate the presence of correlations between the molecular findings and AHO severity. PATIENTS AND METHODS We investigated differential methylation of GNAS regions and STX16 microdeletions in genomic DNA from 40 patients with sporadic AHO and multihormone resistance, with no mutations in Gsalpha-coding GNAS exons. RESULTS Molecular analysis showed GNAS cluster imprinting defects in 24 of the 40 patients analyzed. No STX16 deletion was detected. The presence of imprinting defects was not associated with the severity of AHO or with specific AHO signs. CONCLUSIONS We report the largest series of the literature of patients with clinical AHO and multihormone resistance and no mutation in the Gsalpha gene. Our findings of frequent GNAS imprinting defects further confirm the existence of an overlap between molecular and clinical features of PHP-Ia and PHP-Ib and highlight the necessity of a new clinical classification of the disease that takes into account the recent knowledge on the molecular basis underlying these defects.

Metabolic stress regulates cytoskeletal dynamics and metastasis of cancer cells

Metabolic reprogramming is an important driver of tumor progression; however, the metabolic regulators of tumor cell motility and metastasis are not understood. Here, we show that tumors maintain energy production under nutrient deprivation through the function of HSP90 chaperones compartmentalized in mitochondria. Using cancer cell lines, we found that mitochondrial HSP90 proteins, including tumor necrosis factor receptor-associated protein-1 (TRAP-1), dampen the activation of the nutrient-sensing AMPK and its substrate UNC-51-like kinase (ULK1), preserve cytoskeletal dynamics, and release the cell motility effector focal adhesion kinase (FAK) from inhibition by the autophagy initiator FIP200. In turn, this results in enhanced tumor cell invasion in low nutrients and metastatic dissemination to bone or liver in disease models in mice. Moreover, we found that phosphorylated ULK1 levels were correlated with shortened overall survival in patients with non-small cell lung cancer. These results demonstrate that mitochondrial HSP90 chaperones, including TRAP-1, overcome metabolic stress and promote tumor cell metastasis by limiting the activation of the nutrient sensor AMPK and preventing autophagy.

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells

Summary Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

MicroRNA profiling of hepatocarcinogenesis identifies C19MC cluster as a novel prognostic biomarker in hepatocellular carcinoma

Progressive hepatocarcinogenesis is a stepwise process that drives liver transformation. However, the molecular mechanisms of early liver transformation are far from clear. A role for microRNAs (miRNA) as diagnostic and prognostic factors in human tumours, including hepatocellular carcinoma (HCC), is promising. We aimed to identify novel miRNA as biomarkers for differential diagnosis and predictors of disease progression.

miR-296 regulation of a cell polarity–cell plasticity module controls tumor progression

The expression of small, non-coding RNA or microRNAs (miR), is frequently deregulated in human cancer, but how these pathways affect disease progression is still largely elusive. Here, we report on a miR, miR-296, which is progressively lost during tumor progression and correlates with metastatic disease in colorectal, breast, lung, gastric, parathyroid, liver and bile ducts cancers. Functionally, miR-296 controls a global cell motility gene signature in epithelial cells by transcriptionally repressing the cell polarity–cell plasticity module, Scribble (Scrib). In turn, loss of miR-296 causes aberrantly increased and mislocalized Scrib in human tumors, resulting in exaggerated random cell migration and tumor cell invasiveness. Re-expression of miR-296 in MDA-MB231 cells inhibits tumor growth in vivo. Finally, miR-296 or Scrib levels predict tumor relapse in hepatocellular carcinoma patients. These data identify miR-296 as a global repressor of tumorigenicity and uncover a previously unexplored exploitation of Scrib in tumor progression in humans.

PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion

Significance Despite the promise of personalized cancer medicine, most molecular therapies produce only modest and short-lived patient gains. In addition to drug resistance, it is also possible that tumors adaptively reprogram their signaling pathways to evade therapy-induced “stress” and, in the process, acquire more aggressive disease traits. We show here that small-molecule inhibitors of PI3K, a cancer node and important therapeutic target, induce transcriptional and signaling reprogramming in tumors. This involves the trafficking of energetically active mitochondria to subcellular sites of cell motility, where they provide a potent, “regional” energy source to support tumor cell invasion. Although this response may paradoxically increase the risk of metastasis during PI3K therapy, targeting mitochondrial reprogramming is feasible, and could provide a novel therapeutic strategy. Molecular therapies are hallmarks of “personalized” medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, “spatiotemporal” mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.

The microRNA cluster C19MC is deregulated in parathyroid tumours.

A subset of over-expressed microRNAs (miRNAs) identified in parathyroid carcinomas (Ca) compared to normal glands belongs to C19MC, a cluster on chromosome 19q13.4 involved in stem cell biology and tumourigenesis. In this study, the expression of C19MC-MIR371-3 clusters and the molecular mechanisms presiding their modulation were investigated in a series of six normal parathyroids, 24 adenomas (Ad), 15 Ca and five matched metastases. The general expression levels of C19MC or MIR371-3 clusters in Ad lesions did not differ from normal glands, while they distinguished Ad from Ca at unsupervised hierarchical cluster analysis (P=0.0008). MIR517C showed the most significant difference in expression between Ca and Ad (P=0.0003) and it positively correlated with serum calcium, parathormone and tumour weight. In regard to the molecular mechanism determining C19MC cluster activation, we could detect C19MC copy number (CN) gain in ten Ca (67%) extending distal to the MIR371-3 cluster in almost all samples. Conversely, only four Ad (16%) showed C19MC amplification, with one case presenting distal genomic aberration to MIR371-3. Globally, CN variations of 19q13.4 loci were significantly associated with MIR517C up-regulation (P=0.006). Opposite to normal glands where C19MC promoter was methylated, hypomethylation occurred in 15 out of 30 analysed tumours. Though the epigenetic status did not correlate with C19MC miRNA expression levels, loss of C19MC promoter methylation was significantly associated with Ca and metastatic disease (P=0.01). In conclusion, C19MC cluster aberrations are a characteristic of Ca with respect to Ad. Altogether, these evidences point towards a role for 19q13.4 miRNA clusters as oncogenes in parathyroid tumourigenesis.