Papers by Manuela Lanzafame
Nucleic Acids Research, Jul 27, 2021
Systematic perturbation screens provide comprehensive resources for the elucidation of cancer dri... more Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumorpromoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.
bioRxiv (Cold Spring Harbor Laboratory), Feb 10, 2020
Systematic perturbation screens provide comprehensive resources for the elucidation of cancer dri... more Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumorpromoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.
The EMBO Journal, Aug 19, 2020
Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was init... more Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.
Journal of Controlled Release, Jun 1, 2021
Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcome... more Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.
International Journal of Molecular Sciences, Feb 28, 2018
Whole-transcriptome analyses have revealed that a large proportion of the human genome is transcr... more Whole-transcriptome analyses have revealed that a large proportion of the human genome is transcribed in non-protein-coding transcripts, designated as long non-coding RNAs (lncRNAs). Rather than being "transcriptional noise", increasing evidence indicates that lncRNAs are key players in the regulation of many biological processes, including transcription, post-translational modification and inhibition and chromatin remodeling. Indeed, lncRNAs are widely dysregulated in human cancers, including hepatocellular carcinoma (HCC). Functional studies are beginning to provide insights into the role of oncogenic and tumor suppressive lncRNAs in the regulation of cell proliferation and motility, as well as oncogenic and metastatic potential in HCC. A better understanding of the molecular mechanisms and the complex network of interactions in which lncRNAs are involved could reveal novel diagnostic and prognostic biomarkers. Crucially, it may provide novel therapeutic opportunities to add to the currently limited number of therapeutic options for HCC patients. In this review, we summarize the current status of the field, with a focus on the best characterized dysregulated lncRNAs in HCC.
Initially reported for viral RNA, elongation of polypeptide chains beyond the stop codon (transla... more Initially reported for viral RNA, elongation of polypeptide chains beyond the stop codon (translational readthrough (TR)) also occurs on eukaryotic transcripts. TR diversifies the proteome and can modulate protein levels 1-6. Here we report that AGO1x, a conserved TR isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double stranded RNAs, the induction of the interferon response and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x localizes to the nucleus, in the vicinity of nucleoli. We identify a novel interaction of AGO1x with the Polyribonucleotide Nucleotidyltransferase 1, depletion of either protein leading to dsRNA accumulation and impaired cell proliferation. Our study thus uncovers a novel function of an Argonaute protein outside of the miRNA effector pathway, in buffering dsRNA-induced interferon responses. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study suggests a new direction for limiting tumor growth.
Frontiers in Genetics, Feb 2, 2018
The TP53 gene is the most commonly mutated gene in human cancers and mutations in TP53 have been ... more The TP53 gene is the most commonly mutated gene in human cancers and mutations in TP53 have been shown to have either gain-of-function or loss-of-function effects. Using the data generated by The Cancer Genome Atlas, we sought to define the spectrum of TP53 mutations in hepatocellular carcinomas (HCCs) and their association with clinicopathologic features, and to determine the oncogenic and mutational signatures in TP53-mutant HCCs. Compared to other cancer types, HCCs harbored distinctive mutation hotspots at V157 and R249, whereas common mutation hotspots in other cancer types, R175 and R273, were extremely rare in HCCs. In terms of clinicopathologic features, in addition to the associations with chronic viral infection and high Edmondson grade, we found that TP53 somatic mutations were less frequent in HCCs with cholestasis or tumor infiltrating lymphocytes, but were more frequent in HCCs displaying necrotic areas. An analysis of the oncogenic signatures based on the genetic alterations found in genes recurrently altered in HCCs identified four distinct TP53-mutant subsets, three of which were defined by CTNNB1 mutations, 1q amplifications or 8q24 amplifications, respectively, that co-occurred with TP53 mutations. We also found that mutational signature 12, a liver cancer-specific signature characterized by T>C substitutions, was prevalent in HCCs with wild-type TP53 or with missense TP53 mutations, but not in HCCs with deleterious TP53 mutations. Finally, whereas patients with HCCs harboring deleterious TP53 mutations had worse overall and disease-free survival than patients with TP53-wild-type HCCs, patients with HCCs harboring missense TP53 mutations did not have worse prognosis. In conclusion, our results highlight the importance to consider the genetic heterogeneity among TP53-mutant HCCs in studies of biomarkers and molecular characterization of HCCs.
Annals of Oncology, May 1, 2018
Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of t... more Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine if plasma-derived cellfree DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy. Patients and methods: Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was performed using a panel targeting 46 coding and non-coding genes frequently altered in HCCs. Results: In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations. Conclusion: In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients.
Molecular and Cellular Biology / Genetics, Jul 1, 2018
Commercially available targeted or exome panels miss some genes and regions frequently mutated in... more Commercially available targeted or exome panels miss some genes and regions frequently mutated in hepatocellular carcinoma (HCC); we aim to design and benchmark an HCC-specific panel as a robust high-throughput and cost-effective panel for genomic screening in HCC. We designed an Ion AmpliSeq panel targeting all exons of 33 liver cancer protein-coding genes, recurrently mutated long non-coding RNA genes MALAT1 and NEAT1, the recurrently mutated promoter regions of TERT, WDR74, MED16 and TFPI2, as well as mutation hotspots in 7 additional cancer genes. We profiled DNA from fresh frozen tumor (FFT, n=10, median depth 1495x) and/or formalin-fixed paraffin-embedded (FFPE) tumor with low input DNA (n=36, median depth 530x), and their non-tumoral counterparts from 39 HCCs using the custom panel. We benchmarked the somatic mutations and copy number alterations (CNAs) identified against those from Illumina whole-exome sequencing (WES) of the equivalent FFT samples (median depth 112x). At least one somatic mutation was identified in 35/39 cases using the custom panel. Median of 2.5 (0-74) and 3 (0-76) mutations were identified in FFT and FFPE samples, respectively. 98% (61/62) of the mutations identified from WES of the 10 FFT samples were successfully recovered using the custom panel, with the identification of an additional 6 and 32 mutations in coding and non-coding regions, respectively. Similarly, all 104 mutations identified from WES were also found based on the analysis of our custom panel of the 36 FFPE biopsies. We identified an additional 18 and 70 somatic mutations in coding and non-coding genes, respectively, using our custom panel, including 2 CTNNB1 activating mutations. Furthermore, copy number analysis revealed high correlation (r = 0.8) in the identification of gains/ amplifications and losses/ homozygous deletions in the genes included in the custom-panel between our custom panel and the WES. Here, we develop a robust high-throughput and cost-effective tool for HCC genomic screening that allows the identification with high sensitivity and specificity of somatic mutations as well as copy number alterations in routine diagnostic tissue specimens. Citation Format: Viola Paradiso, Andrea Garofoli, Nadia Tosti, Valeria Perrina, Manuela Lanzafame, Luca Quagliata, Matthias S. Matter, Stefan Wieland, Markus H. Heim, Salvatore Piscuoglio, Charlotte K. Ng, Luigi M. Terracciano. Diagnostic targeted sequencing panel for hepatocellular carcinoma genomic screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 433.
Melanoma Research, Aug 1, 2008
The incidence of melanoma has dramatically increased in many countries (it is 4.5 cases every 100... more The incidence of melanoma has dramatically increased in many countries (it is 4.5 cases every 100 000 inhabitants in Sicily) and Xq27 region contains genes important in cancer like the SPANX (sperm protein associated with the nucleus in the X chromosome) gene family. These genes, made up of two exons separated by an intron of about 650 base pair, are expressed in sperm cells and in many tumours, including melanoma. These observations suggested that SPANX genes, or some of them, may be involved in melanoma development. The aim of this study was to investigate the genetic variability of SPANX-B and SPANX-C in a sample of Sicilian male population including patients with melanoma of the skin and controls. A total of 99 patients were enrolled in this study. They included: 17 male patients with cutaneous melanoma and 82 normal males. Semiquantitative fluorescent multiplex PCR dosage analysis was carried out to identify the variety of classes of SPANX-B and SPANX-C genes. Sixteen and 13 genetic classes were detected for SPANX-B and SPANX-C genes, respectively. A statistical significant difference for a particular class of SPANX-C gene was found comparing patients with melanoma and controls (P = 0.011). Further investigations should be conducted to confirm these observations and to evaluate the possible implication of other genes of the region Xq27-28 in melanoma.
Oncotarget, 2015
The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP... more The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches.
The American Journal of Human Genetics, 2016
The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA pol... more The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEb). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEa and TFIIEb) as well as decreased phosphorylation of TFIIEa in cells from both children. Interestingly, decreased phosphorylation of TFIIEa was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.
Nucleic Acids Research, 2021
CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER)... more CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients resu...
Annals of Oncology
Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of t... more Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine if plasma-derived cellfree DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy. Patients and methods: Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was performed using a panel targeting 46 coding and non-coding genes frequently altered in HCCs. Results: In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations. Conclusion: In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients.
Nucleic Acids Research, 2021
Systematic perturbation screens provide comprehensive resources for the elucidation of cancer dri... more Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/β-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by ...
Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcome... more Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressi...
BMC Complementary Medicine and Therapies
Background Hyperactivation of mechanistic target of rapamycin (mTOR) signaling pathway is involve... more Background Hyperactivation of mechanistic target of rapamycin (mTOR) signaling pathway is involved in the regulation of cellular growth, proliferation, and more in general, is a common phenomenon in most types of cancers. Thus, natural substances targeting this pathway can be of great therapeutic potential in supporting the treatment of tumor patients. Rhus tripartita (Ucria) Grande is a plant growing in desertic areas which is traditionally used for the treatment of several diseases in Tunisia. In the present work, the biochemical profile of the main compounds present in the plant leaf extract was determined and the anti-leukemic potential of the plant extracts against acute monocytic leukaemia (AML) THP-1 cells was investigated. Methods After HPLC identification of some phenolic compounds present in the plant extract and the quantification of saponin content, the cytotoxic effect of Rhus tripartita extracts on THP-1 cell culture was evaluated using the colorimetric MTT assay for c...
The EMBO Journal
Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was init... more Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein‐level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double‐stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA‐induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.
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Papers by Manuela Lanzafame