Carmelo Laudanna

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.

Cancer-Associated Immune Resistance and Evasion of Immune Surveillance in Colorectal Cancer

Data from molecular profiles of tumors and tumor associated cells provide a model in which cancer cells can acquire the capability of avoiding immune surveillance by expressing an immune-like phenotype. Recent works reveal that expression of immune antigens (PDL1, CD47, CD73, CD14, CD68, MAC387, CD163, DAP12, and CD15) by tumor cells “immune resistance,” combined with prometastatic function of nonmalignant infiltrating cells, may represent a strategy to overcome the rate-limiting steps of metastatic cascade through (a) enhanced interactions with protumorigenic myeloid cells and escape from T-dependent immune response mediated by CD8+ and natural killer (NK) cells; (b) production of immune mediators that establish a local and systemic tumor-supportive environment (premetastatic niche); (c) ability to survive either in the peripheral blood as circulating tumor cells (CTCs) or at the metastatic site forming a cooperative prometastatic loop with foreign “myeloid” cells, macrophages, and neutrophils, respectively. The development of cancer-specific “immune resistance” can be orchestrated either by cooperation with tumor microenvironment or by successive rounds of genetic/epigenetic changes. Recognition of the applicability of this model may provide effective therapeutic avenues for complete elimination of immune-resistant metastatic cells and for enhanced antitumor immunity as part of a combinatorial strategy.

Analysis of miRNA profiles identified miR-196a as a crucial mediator of aberrant PI3K/AKT signaling in lung cancer cells

Hyperactivation of the PI3K/AKT pathway is observed in most human cancer including lung carcinomas. Here we have investigated the role of miRNAs as downstream targets of activated PI3K/AKT signaling in Non Small Cell Lung Cancer (NSCLC). To this aim, miRNA profiling was performed in human lung epithelial cells (BEAS-2B) expressing active AKT1 (BEAS-AKT1-E17K), active PI3KCA (BEAS-PIK3CA-E545K) or with silenced PTEN (BEAS-shPTEN). Twenty-four differentially expressed miRNAs common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells were identified through this analysis, with miR-196a being the most consistently up-regulated miRNA. Interestingly, miR-196a was significantly overexpressed also in human NSCLC-derived cell lines (n=11) and primary lung cancer samples (n=28). By manipulating the expression of miR-196a in BEAS-2B and NCI-H460 cells, we obtained compelling evidence that this miRNA acts downstream the PI3K/AKT pathway, mediating some of the proliferative, pro-migratory and tumorigenic activity that this pathway exerts in lung epithelial cells, possibly through the regulation of FoxO1, CDKN1B (hereafter p27) and HOXA9.

Aberrant BLM cytoplasmic expression associates with DNA damage stress and hypersensitivity to DNA-damaging agents in colorectal cancer

BackgroundBloom syndrome is a rare and recessive disorder characterized by loss-of-function mutations of the BLM gene, which encodes a RecQ 3′–5′ DNA helicase. Despite its putative tumor suppressor function, the contribution of BLM to human sporadic colorectal cancer (CRC) remains poorly understood.MethodsThe transcriptional regulation mechanism underlying BLM and related DNA damage response regulation in independent CRC subsets and a panel of derived cell lines was investigated by bioinformatics analysis, the transcriptomic profile, a CpG island promoter methylation assay, Western blot, and an immunolocalization assay.ResultsIn silico analysis of gene expression data sets revealed that BLM is overexpressed in poorly differentiated CRC and exhibits a close connection with shorter relapse-free survival even after adjustment for prognostic factors and pathways that respond to DNA damage response through ataxia telangiectasia mutated (ATM) signaling. Functional characterization demonstrated that CpG island promoter hypomethylation increases BLM expression and associates with cytoplasmic BLM mislocalization and increased DNA damage response both in clinical CRC samples and in derived cancer cell lines. The DNA-damaging agent S-adenosylmethionine suppresses BLM expression, leading to the inhibition of cell growth following accumulation of DNA damage. In tumor specimens, cytoplasmic accumulation of BLM correlates with DNA damage and γH2AX and phosphorylated ATM foci and predicts long-term progression-free survival in metastatic patients treated with irinotecan.ConclusionsTaken together, the findings of this study provide the first evidence that cancer-linked DNA hypomethylation and cytosolic BLM mislocalization might reflect compromised levels of DNA-repair activity and enhanced hypersensitivity to DNA-damaging agents in CRC patients.

Identification of different mutational profiles in cancers arising in specific colon segments by next generation sequencing

The objective of this study was to investigate the mutational profiles of cancers arising in different colon segments. To this aim, we have analyzed 37 colon cancer samples by use of the Ion AmpliSeq™ Comprehensive Cancer Panel. Overall, we have found 307 mutated genes, most of which already implicated in the development of colon cancer. Among these, 15 genes were mutated in tumors originating in all six colon segments and were defined “common genes” (i.e. APC, PIK3CA, TP53) whereas 13 genes were preferentially mutated in tumors originating only in specific colon segments and were defined “site-associated genes” (i.e. BLNK, PTPRD). In addition, the presence of mutations in 10 of the 307 identified mutated genes (NBN, SMUG1, ERBB2, PTPRT, EPHB1, ALK, PTPRD, AURKB, KDR and GPR124) were found to be of clinical relevance. Among clinically relevant genes, NBN and SMUG1 were identified as independent prognostic factors that predicted poor survival in colon cancer patients. In conclusion, the findings reported here indicate that tumors arising in different colon segments present differences in the type and/or frequency of genetic variants, with two of them being independent prognostic factors that predict poor survival in colon cancer patients.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

Identification of copy number alterations in colon cancer from analysis of amplicon-based Next generation sequencing data

The objective of this study was to determine the feasibility to detect copy number alterations in colon cancer samples using Next Generation Sequencing data and to elucidate the association between copy number alterations in specific genes and the development of cancer in different colon segments. We report the successful detection of somatic changes in gene copy number in 37 colon cancer patients by analysis of sequencing data through Amplicon CNA Algorithm. Overall, we have found a total of 748 significant copy number alterations in 230 significant genes, of which 143 showed CN losses and 87 showed CN gains. Validation of results was performed on 20 representative genes by quantitative qPCR and/or immunostaining. By this analysis, we have identified 4 genes that were subjected to copy number alterations in tumors arising in all colon segments (defined “common genes”) and the presence of copy number alterations in 14 genes that were significantly associated to one specific site (defined “site-associated genes”). Finally, copy number alterations in ASXL1, TSC1 and IL7R turned out to be clinically relevant since the loss of TSC1 and IL7R was associated with advanced stages and/or reduced survival whereas copy number gain of ASXL1 was associated with good prognosis.

Centrosome Linker–induced Tetraploid Segregation Errors Link Rhabdoid Phenotypes and Lethal Colorectal Cancers

Centrosome anomalies contribute to tumorigenesis, but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAFV600E-mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosome linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAFV600E-mutant and microsatellite stable (MSS) rhabdoid colorectal cancers, but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAFV600E-mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors, resulting in a highly aggressive rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability. Implications: Mis-segregation of chromosomes is a prominent feature of chromosome instability and intratumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. This study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Mol Cancer Res; 16(9); 1385–95. ©2018 AACR.

Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

BackgroundImprovement in genetic characterization of Colon Cancer (CC) patients is required to propose new potential targets, since surgical resection coupled to chemotherapy, presents several limits such as cancer recurrence and drug resistance. Targeted therapies have more efficacy and less toxicity than standard treatments. One of the most relevant cancer-specific actionable targets are receptor tyrosine kinases (RTKs) whose role in CC need to be better investigated.MethodsWe have analysed 37 CC patients using the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP). We have confirmed the somatic nature of RET variants through Sanger sequencing and assessed RET activation status and protein expression by immunofluorescence and western-blot analyses. We have used RET mutant expression vectors to evaluate the effect of selected mutations in HEK293 cells by performing proliferation, migration and clonogenic assays.ResultsAmong the 409 cancer-related genes included in the CCP we have focused on the RTKs. Overall, we have observed 101 different potentially damaging variants distributed across 31 RTK genes in 28 patients. The most frequently mutated RTKs were FLT4, ROS1, EPH7, ERBB2, EGFR, RET, FGFR3 and FGFR4. In particular, we have identified 4 different somatic variants in 10% of CC patients in RET proto-oncogene. Among them, we have demonstrated that the G533C variant was able to activate RET by promoting dimer formation and enhancing Y1062 phosphorylation. Moreover, we have demonstrated that RET G533C variant was able to stimulate anchorage-dependent proliferation, migration and clonogenic cell survival. Notably, the effects induced by the RET G533C variant were abolished by vandetanib.ConclusionsThe discovery of pathogenic variants across RTK genes in 75% of the CC patients under analysis, suggests a previously underestimated role for RTKs in CC development. The identification of a gain-of-function RET mutation in CC highlights the potential use of RET in targeted therapy.

Genetic characterization of colorectal cancer using a next generation sequencing approach to identify a specific pattern of somatic alterations in tumors arising from different anatomic sites

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide, with nearly 1.4 million new cases diagnosed in 2012. CRC results from the accumulation of multiple genetic and epigenetic aberrations. Tumor localization in the large intestine tract determines different surgical approaches and treatment options. Considering the heterogeneous nature of these tumors we hypothesized that different patterns of molecular alterations could be associated with a specific anatomical location.