Carlo Capalbo

PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress

The Hedgehog (Hh) signaling regulates tissue development, and its aberrant activation is a leading cause of malignancies, including medulloblastoma (Mb). Hh-dependent tumorigenesis often occurs in synergy with other mechanisms, such as loss of p53, the master regulator of the DNA damage response. To date, little is known about mechanisms connecting DNA-damaging events to morphogen-dependent processes. Here, we show that genotoxic stress triggers a cascade of signals, culminating with inhibition of the activity of Gli1, the final transcriptional effector of Hh signaling. This inhibition is dependent on the p53-mediated elevation of the acetyltransferase p300/CBP-associated factor (PCAF). Notably, we identify PCAF as a novel E3 ubiquitin ligase of Gli1. Indeed PCAF, but not a mutant with a deletion of its ubiquitination domain, represses Hh signaling in response to DNA damage by promoting Gli1 ubiquitination and its proteasome-dependent degradation. Restoring Gli1 levels rescues the growth arrest and apoptosis effect triggered by genotoxic drugs. Consistently, DNA-damaging agents fail to inhibit Gli1 activity in the absence of either p53 or PCAF. Finally, Mb samples from p53-null mice display low levels of PCAF and upregulation of Gli1 in vivo, suggesting PCAF as potential therapeutic target in Hh-dependent tumors. Together, our data define a mechanism of inactivation of a morphogenic signaling in response to genotoxic stress and unveil a p53/PCAF/Gli1 circuitry centered on PCAF that limits Gli1-enhanced mitogenic and prosurvival response.

microRNA‐17‐92 cluster is a direct Nanog target and controls neural stem cell through Trp53inp1

The transcription factor Nanog plays a critical role in the self‐renewal of embryonic stem cells as well as in neural stem cells (NSCs). microRNAs (miRNAs) are also involved in stemness regulation. However, the miRNA network downstream of Nanog is still poorly understood. High‐throughput screening of miRNA expression profiles in response to modulated levels of Nanog in postnatal NSCs identifies miR‐17‐92 cluster as a direct target of Nanog. Nanog controls miR‐17‐92 cluster by binding to the upstream regulatory region and maintaining high levels of transcription in NSCs, whereas Nanog/promoter association and cluster miRNAs expression are lost alongside differentiation. The two miR‐17 family members of miR‐17‐92 cluster, namely miR‐17 and miR‐20a, target Trp53inp1, a downstream component of p53 pathway. To support a functional role, the presence of miR‐17/20a or the loss of Trp53inp1 is required for the Nanog‐induced enhancement of self‐renewal of NSCs. We unveil an arm of the Nanog/p53 pathway, which regulates stemness in postnatal NSCs, wherein Nanog counteracts p53 signals through miR‐17/20a‐mediated repression of Trp53inp1.

Prevalence of BRCA1 and BRCA2 genomic rearrangements in a cohort of consecutive Italian breast and/or ovarian cancer families

Germline point mutations in BRCA1 and BRCA2 genes account for about 30% of the inherited breast and ovarian cancers. Germline genomic rearrangements have been found in both BRCA1 and BRCA2 genes, but the extent to which these alterations might contribute to increasing the actual mutation detection rate is still debated. Here we screened a cohort of 112 consecutive Italian families at moderate-to-high risk for breast and/or ovarian cancer for BRCA1 and BRCA2 point mutations and genomic rearrangements. Of the 83 point mutation negative probands, two (2.4%) showed BRCA1 rearrangements, accounting for 10.5% of the BRCA1 mutations. BRCA1 del18–19 has been previously described in another Italian family, while the molecular characterization of the BRCA1 del23–24 is given here for the first time. Conversely, we failed to identify any BRCA2 rearrangements even in the hereditary breast cancer families, where we detected an higher prevalence of BRCA2 compared to BRCA1 point mutations. Our results support the idea that search for BRCA1 rearrangements should be included in the genetic screening of even moderate risk breast/ovarian cancer families. In contrast, they suggest BRCA2 rearrangements might be very rare out of the high risk families including a male breast cancer.

High-throughput microRNA profiling of pediatric high-grade gliomas

Background High-grade gliomas (HGGs) account for 15% of all pediatric brain tumors and are a leading cause of cancer-related mortality and morbidity. Pediatric HGGs (pHGGs) are histologically indistinguishable from their counterpart in adulthood. However, recent investigations indicate that differences occur at the molecular level, thus suggesting that the molecular path to gliomagenesis in childhood is distinct from that of adults. MicroRNAs (miRNAs) have been identified as key molecules in gene expression regulation, both in development and in cancer. miRNAs have been investigated in adult high-grade gliomas (aHGGs), but scant information is available for pHGGs. Methods We explored the differences in microRNAs between pHGG and aHGG, in both fresh-frozen and paraffin-embedded tissue, by high-throughput miRNA profiling. We also evaluated the biological effects of miR-17-92 cluster silencing on a pHGG cell line. Results Comparison of miRNA expression patterns in formalin versus frozen specimens resulted in high correlation between both types of samples. The analysis of miRNA profiling revealed a specific microRNA pattern in pHGG with an overexpression and a proliferative role of the miR-17-92 cluster. Moreover, we highlighted a possible quenching function of miR-17-92 cluster on its target gene PTEN, together with an activation of tumorigenic signaling such as sonic hedgehog in pHGG. Conclusions Our results suggest that microRNA profiling represents a tool to distinguishing pediatric from adult HGG and that miR-17-92 cluster sustains pHGG.

Novel BRCA1 and BRCA2 germline mutations and assessment of mutation spectrum and prevalence in Italian breast and/or ovarian cancer families.

Familial aggregations of breast/ovarian cancer cases frequently depend on BRCA1/2 pathogenic mutations. Here we counselled 120 Italian breast/ovarian cancer families and selected 73 probands for BRCA1/2 mutation screening. Through this analysis we defined the prevalence of BRCA1/2 pathogenic mutations occurring in Italian breast/ovarian cancer families, enlarged the spectrum of Italian BRCA1/2 mutations by 15% and report on the identification of 13 novel variants, including two deleterious truncating mutations and two potentially pathogenic missense mutations, on the BRCA1 and BRCA2 genes. Finally in hereditary breast cancer families with three or more female breast cancer cases we observed a low mutation prevalence and a significant association with BRCA2 mutations.

The role of peroxiredoxins in cancer (Review)

Peroxiredoxins (PRDXs) are a ubiquitously expressed family of small (22–27 kDa) non-seleno peroxidases that catalyze the peroxide reduction of H2O2, organic hydroperoxides and peroxynitrite. They are highly involved in the control of various physiological functions, including cell growth, differentiation, apoptosis, embryonic development, lipid metabolism, the immune response, as well as cellular homeostasis. Although the protective role of PRDXs in cardiovascular and neurological diseases is well established, their role in cancer remains controversial. Increasing evidence suggests the involvement of PRDXs in carcinogenesis and in the development of drug resistance. Numerous types of cancer cells, in fact, are characterized by an increase in reactive oxygen species (ROS) production, and often exhibit an altered redox environment compared with normal cells. The present review focuses on the complex association between oxidant balance and cancer, and it provides a brief account of the involvement of PRDXs in tumorigenesis and in the development of chemoresistance.

Galectin-3 is a marker of favorable prognosis and a biologically relevant molecule in neuroblastic tumors

Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a β-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P<0.001) and Shimada favorable phenotype (P=0.001), but not with other prognostically relevant features. Importantly, Gal-3 expression was associated with a better 5-year overall survival (P=0.003), and with improved cumulative survival in patient subsets at worse prognosis, such as older age at diagnosis, advanced stages or NB histopathological classification. In vitro, Gal-3 expression and nuclear accumulation accompanied retinoic acid-induced cell differentiation in NB cell lines. Forced Gal-3 overexpression increased phenotypic differentiation and substrate adherence, while inhibiting proliferation. Altogether, these findings suggest that Gal-3 is a biologically relevant player for neuroblastic tumors, whose determination by conventional immunohistochemistry might be used for outcome assessment and patient’s risk stratification in the clinical setting.

Vemurafenib and panitumumab combination tailored therapy in BRAF-mutated metastatic colorectal cancer: A case report

As the knowledge on cancer genetic alterations progresses, it fosters the need for more personalized therapeutic intervention in modern cancer management. Recently, mutations in KRAS, BRAF, and PIK3CA genes have emerged as important mechanisms of resistance to EGFR-targeted therapy in metastatic colorectal cancer (mCRC). Here we report the first case of a mCRC patient whose disease had progressed on standard lines of treatment and for which we devised a personalized therapeutic approach consisting of vemurafenib (ZelborafTM) and panitumumab (VectibixTM), based on the following molecular profile: BRAFV600E-mutant, amplified EGFR (double positive) and WT KRAS, WT PIK3CA, not-amplified HER2 (triple negative). This new combination therapy was well tolerated and resulted in a strong control of the disease. In particular, the vemurafenib-panitumumab combination appears to limit the typical toxicity of single agents, since no cutaneous toxic effects typically associated with vemurafenib were observed. Here we report the first clinical evidence that the combination of an anti-EGFR (panitumumab) and an inhibitor of BRAFV600E (vemurafenib) is well tolerated and results in a strong disease control in an extensively pretreated mCRC patient.

Validation of the Ion Torrent PGM sequencing for the prospective routine molecular diagnostic of colorectal cancer.

OBJECTIVES Treatment individualization based on specific molecular biomarkers is becoming increasingly important in oncology. In colorectal cancer (CRC), the molecular characterization of RAS and BRAF mutation status for prognostic and predictive purposes is commonly performed by different validated methods. However, as the number of clinically relevant mutations to be analyzed increases, the definition of new approaches for more sensitive, rapid and economic patient selection urges. To this aim, we evaluated the Ion Semiconductor sequencing using the Ion Torrent Personal Genome Machine (IT-PGM) in our routine molecular diagnostics for CRC in comparison with the gold standard direct Sanger sequencing. DESIGN AND METHODS Formalin-fixed and paraffin-embedded tumor tissues obtained by surgery or biopsy of 66 CRCs were collected. DNA was extracted and sequenced by IT-PGM and Sanger method. RESULTS The proposed IT-PGM sequencing strategy exceeded the 500 reads of coverage for all clinically relevant RAS/BRAF amplicons in most samples and thus guaranteed optimal determination. Indeed, the frequencies and the mutational spectrum of RAS and BRAF mutations were in agreement with literature data and revealed 100% concordance between the IT-PGM and routine Sanger sequencing approaches. Turnaround time and cost evaluation indicate that the IT-PGM sequencing permits the characterization of the clinically relevant mutational spots at lower cost and turnaround time compared to Sanger sequencing and allows inclusion of additional amplicons whose characterization may acquire significance in the very next future. CONCLUSION The IT-PGM is a valid, flexible, sensitive and economical method alternative to the Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor therapy for metastatic CRC.

PRDX1 and PRDX6 are repressed in papillary thyroid carcinomas via BRAF V600E-dependent and -independent mechanisms

Many clinical studies highlight the dichotomous role of PRDXs in human cancers, where they can exhibit strong tumor-suppressive or tumor-promoting functions. Recent evidence suggests that lower expression of PRDXs correlates with cancer progression in colorectal cancer (CRC) or in esophageal squamous carcinoma. In the thyroid, increased levels of PRDX1 has been described in follicular adenomas and carcinomas, as well as in thyroiditis, while reduced levels of PRDX6 has been found in follicular adenomas. We studied the expression of PRDX1 and PRDX6, in a series of thyroid tissue samples, covering different thyroid diseases, including 13 papillary thyroid carcinomas (PTCs). Our results show that PRDX1 and PRDX6 are significantly reduced in all PTCs compared to normal tissues, to non-neoplastic tissue (MNG) or follicular neoplasms. This reduction is strongly evident in PTCs harboring BRAF V600E (31% of our cases). Using TPC-1 and BCPAP and FRTL-5 cell lines, we demonstrate for the first time that the presence of BRAF V600E is responsible of the hypoexpression of PRDX1 and PRDX6 both at mRNA and protein levels. Finally, independently of BRAF status, we observe an interesting correlation between the tumor size, the presence of lymph node metastasis and the lowest PRDX1 and PRDX6 levels. Therefore, these data indicate that PRDX1 and PRDX6 expression not only may play a key role in papillary thyroid carcinogenesis via a BRAF V600E-dependent mechanism, but their determination could be considered as potential tumor marker for indicating tumor progression in PTCs, independently of BRAF status.