RESUMEN
Juvenile polyposis syndrome (JPS) is an inherited autosomal dominant condition that predisposes to the development of juvenile polyps throughout the gastrointestinal (GI) tract, and it poses an increased risk of GI malignancy. Germline causative variants were identified in the SMAD4 gene in a subset (20%) of JPS cases. Most SMAD4 germline genetic variants published to date are missense, nonsense, and frameshift mutations. SMAD4 germline alterations predicted to result in aberrant splicing have rarely been reported. Here, we report two unrelated Italian families harboring two different SMAD4 intronic variants, c.424+5G>A and c.425-9A>G, which are clinically associated with colorectal cancer and/or juvenile GI polyps. In silico prediction analysis, in vitro minigene assays, and RT-PCR showed that the identified variants lead to aberrant SMAD4 splicing via the exonization of intronic nucleotides, resulting in a premature stop codon. This is expected to cause the production of a truncated protein. This study expands the landscape of SMAD4 germline genetic variants associated with GI polyposis and/or cancer. Moreover, it emphasizes the importance of the functional characterization of SMAD4 splicing variants through RNA analysis, which can provide new insights into genetic disease variant interpretation, enabling tailored genetic counseling, management, and surveillance of patients with GI polyposis and/or cancer.
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Poliposis Intestinal , Síndromes Neoplásicos Hereditarios , Empalme del ARN , Proteína Smad4 , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación de Línea Germinal , Poliposis Intestinal/genética , Poliposis Intestinal/congénito , Intrones/genética , Síndromes Neoplásicos Hereditarios/genética , Linaje , Empalme del ARN/genética , Proteína Smad4/genéticaRESUMEN
BACKGROUND: SMYD3 has been found implicated in cancer progression. Its overexpression correlates with cancer growth and invasion, especially in gastrointestinal tumors. SMYD3 transactivates multiple oncogenic mechanisms, favoring cancer development. Moreover, it was recently shown that SMYD3 is required for DNA restoration by promoting homologous recombination (HR) repair. METHODS: In cellulo and in vivo models were employed to investigate the role of SMYD3 in cancer chemoresistance. Analyses of SMYD3-KO cells, drug-resistant cancer cell lines, patients' residual gastric or rectal tumors that were resected after neoadjuvant therapy and mice models were performed. In addition, the novel SMYD3 covalent inhibitor EM127 was used to evaluate the impact of manipulating SMYD3 activity on the sensitization of cancer cell lines, tumorspheres and cancer murine models to chemotherapeutics (CHTs). RESULTS: Here we report that SMYD3 mediates cancer cell sensitivity to CHTs. Indeed, cancer cells lacking SMYD3 functions showed increased responsiveness to CHTs, while restoring its expression promoted chemoresistance. Specifically, SMYD3 is essential for the repair of CHT-induced double-strand breaks as it methylates the upstream sensor ATM and allows HR cascade propagation through CHK2 and p53 phosphorylation, thereby promoting cancer cell survival. SMYD3 inhibition with the novel compound EM127 showed a synergistic effect with CHTs in colorectal, gastric, and breast cancer cells, tumorspheres, and preclinical colorectal cancer models. CONCLUSIONS: Overall, our results show that targeting SMYD3 may be an effective therapeutic strategy to overcome chemoresistance.
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Daño del ADN , Reparación del ADN , Resistencia a Antineoplásicos , N-Metiltransferasa de Histona-Lisina , Humanos , Animales , Ratones , Reparación del ADN/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , FemeninoRESUMEN
BACKGROUND: Activation of the Wnt pathway has been linked to colorectal cancer (CRC). Previous reports suggest that Wnt3a can activate p38. Besides, p38α feeds into the canonical Wnt/ß-catenin pathway by inhibiting GSK3ß through phosphorylation. Recently, we identified p38α as a new druggable member of ß-catenin chromatin-associated kinase complexes in CRC. METHODS: The functional relationship between p38α and ß-catenin was characterized in CRC cells, patient-derived CRC stem cells, patient-derived tumor intestinal organoids, and in vivo models (C57BL/6-APCMin/+ mice). The role of p38α in ß-catenin transcriptional activity was assessed by pharmacological inhibition with ralimetinib. RESULTS: We used the GSK3ß inhibitor TWS-119, which promotes the activation of Wnt signaling, to uncouple p38α nuclear/cytoplasmatic functions in the Wnt pathway. Upon GSK3ß inhibition, nuclear p38α phosphorylates ß-catenin at residues S111 and T112, allowing its binding to promoter regions of Wnt target genes and the activation of a transcriptional program implicated in cancer progression. If p38α is pharmacologically inhibited in addition to GSK3ß, ß-catenin is prevented from promoting target gene transcription, which is expected to impair carcinogenesis. CONCLUSIONS: p38α seems to play a dual role as a member of the ß-catenin destruction complex and as a ß-catenin chromatin-associated kinase in CRC. This finding may help elucidate mechanisms contributing to human colon tumor pathogenesis and devise new strategies for personalized CRC treatment.
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Classic galactosemia is an autosomal recessive inherited liver disorder of carbohydrate metabolism caused by deficient activity of galactose-1-phosphate uridylyltransferase (GALT). While a galactose-restricted diet is lifesaving, most patients still develop long-term complications. In this study, we report on a two-week-old female patient who is a compound heterozygote for a known pathogenic variant (p.K285N) and a novel missense variant (p.A303D) in the GALT gene. Segregation analysis showed that the patient inherited the p.K285N pathogenic variant from her father and the p.A303D variant from her mother. A bioinformatics analysis to predict the impact of the p.A303D missense variant on the structure and stability of the GALT protein revealed that it may be pathogenic. Based on this finding, we performed a literature review of all GALT missense variants identified in homozygous and compound heterozygous galactosemia patients carrying the p.K285N pathogenic variant to explore their molecular effects on the clinical phenotype of the disease. Our analysis revealed that these missense variants are responsible for a wide range of molecular defects. This study expands the clinical and mutational spectrum in classic galactosemia and reinforces the importance of understanding the molecular consequences of genetic variants to incorporate genetic analysis into clinical care.
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Galactosemias , UTP-Hexosa-1-Fosfato Uridililtransferasa , Femenino , Humanos , Galactosa , Galactosemias/genética , Mutación , Mutación Missense , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismoRESUMEN
Cells respond to DNA damage by activating a complex array of signaling networks, which include the AMPK and mTOR pathways. After DNA double-strand breakage, ATM, a core component of the DNA repair system, activates the AMPK-TSC2 pathway, leading to the inhibition of the mTOR cascade. Recently, we showed that both AMPK and mTOR interact with SMYD3, a methyltransferase involved in DNA damage response. In this study, through extensive molecular characterization of gastrointestinal and breast cancer cells, we found that SMYD3 is part of a multiprotein complex that is involved in DNA damage response and also comprises AMPK and mTOR. In particular, upon exposure to the double-strand break-inducing agent neocarzinostatin, SMYD3 pharmacological inhibition suppressed AMPK cascade activation and thereby promoted the mTOR pathway, which reveals the central role played by SMYD3 in the modulation of AMPK-mTOR signaling balance during cancer cell response to DNA double-strand breaks. Moreover, we found that SMYD3 can methylate AMPK at the evolutionarily conserved residues Lys411 and Lys424. Overall, our data revealed that SMYD3 can act as a bridge between the AMPK and mTOR pathways upon neocarzinostatin-induced DNA damage in gastrointestinal and breast cancer cells.
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Neoplasias de la Mama , Cinostatina , Humanos , Femenino , Proteínas Quinasas Activadas por AMP/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Daño del ADN , ADN , N-Metiltransferasa de Histona-Lisina/genéticaRESUMEN
SMDY3 is a histone-lysine N-methyltransferase involved in several oncogenic processes and is believed to play a major role in various cancer hallmarks. Recently, we identified ATM, BRCA2, CHK2, MTOR, BLM, MET, AMPK, and p130 as direct SMYD3 interactors by taking advantage of a library of rare tripeptides, which we first tested for their in vitro binding affinity to SMYD3 and then used as in silico probes to systematically search the human proteome. Here, we used this innovative approach to identify further SMYD3-interacting proteins involved in crucial cancer pathways and found that the chromatin remodeling factors EP300 and TRRAP interact directly with SMYD3, thus linking SMYD3 to the emerging 'nonmutational epigenetic reprogramming' cancer hallmark. Of note, we validated these interactions in gastrointestinal cancer cell lines, including HCT-116 cells, which harbor a C-terminal truncating mutation in EP300, suggesting that EP300 binds to SMYD3 via its N-terminal region. While additional studies are required to ascertain the functional mechanisms underlying these interactions and their significance, the identification of two novel SMYD3 interactors involved in epigenetic cancer hallmark pathways adds important pieces to the puzzle of how SMYD3 exerts its oncogenic role.
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Gastric cancer (GC) is the third most deadly cancer worldwide. Considerable efforts have been made to find targetable drivers in order to improve patient outcomes. MET is one of the most important factors involved in GC initiation and progression as it plays a major role in GC invasiveness and is related to cancer stemness. Unfortunately, treatment strategies targeting MET are still limited, with a proportion of patients responding to therapy but later developing resistance. Here, we showed that MET is a molecular partner of the SMYD3 methyltransferase in GC cells. Moreover, we found that SMYD3 pharmacological inhibition affects the HGF/MET downstream signaling pathway. Extensive cellular analyses in GC models indicated that EM127, a novel active site-selective covalent SMYD3 inhibitor, can be used as part of a synergistic approach with MET inhibitors in order to enhance the targeting of the HGF/MET pathway. Importantly, our data were confirmed in a 3D GC cell culture system, which was used as a surrogate to evaluate stemness characteristics. Our findings identify SMYD3 as a promising therapeutic target to impair the HGF/MET pathway for the treatment of GC.
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Neoplasias Gástricas , Humanos , Neoplasias Gástricas/metabolismo , Transducción de Señal , Factor de Crecimiento de Hepatocito , N-Metiltransferasa de Histona-Lisina/metabolismoRESUMEN
Lynch syndrome (LS) is an inherited cancer susceptibility syndrome caused by germline mutations in a DNA mismatch repair (MMR) gene or in the EPCAM gene. LS is associated with an increased lifetime risk of colorectal cancer (CRC) and other malignancies. The screening algorithm for LS patient selection is based on the identification of CRC specimens that have MMR loss/high microsatellite instability (MSI-H) and are wild-type for BRAFV600. Here, we sought to clinically and molecularly characterize patients with these features. From 2017 to 2023, 841 CRC patients were evaluated for MSI and BRAFV600E mutation status, 100 of which showed MSI-H. Of these, 70 were wild-type for BRAFV600. Among these 70 patients, 30 were genetically tested for germline variants in hereditary cancer predisposition syndrome genes. This analysis showed that 19 of these 30 patients (63.3%) harbored a germline pathogenic or likely pathogenic variant in MMR genes, 2 (6.7%) harbored a variant of unknown significance (VUS) in MMR genes, 3 (10%) harbored a VUS in other cancer-related genes, and 6 (20%) were negative to genetic testing. These findings highlight the importance of personalized medicine for tailored genetic counseling, management, and surveillance of families with LS and other hereditary cancer syndromes.
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Nowadays, different systems for reducing pesticides in table grapes are being tested at different production stages either in the field or in postharvest. The present study tested ozonated water treatments at the beginning of the cold storage of the Princess® seedless table grape variety to reduce the residue contents of some pesticides and to evaluate their effect on gray mold and the berry microbiome. An ozone generator capable of producing an ozone concentration ranging from 18 to 65 Nm3 was utilized for obtaining three ozone concentration levels in water: 3, 5 and 10 mg/L. Ozonated water was placed in a 70 L plastic box where 500 g grape samples closed in perforated plastic clamshell containers were immersed utilizing two washing times (5 and 10 min). Overall, six ozonated water treatments were tested. After the ozonated water treatments, all samples were stored for 30 days at 2 °C and 95% relative humidity to simulate commercial practices. The pesticide residue contents were determined before the ozonated water treatments (T0) and 30 days after the cold storage (T1). The treatments with ozonated water washing reduced the pesticide residues up to 100%, while the SO2 control treatment reduced the pesticide residues ranging from 20.7 to 60.7%. Using 3 mg/L ozonated water to wash grapes for 5 min represented the optimal degradation conditions for all of the analyzed pesticides, except for fludioxonil, which degraded better with a washing time of 10 min. The ozone treatments did not significantly reduce the gray mold and the fungal and bacterial microbiome, while a relevant reduction was observed in the yeast population.
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Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies worldwide. While population-wide screening recommendations for PDAC in asymptomatic individuals are not achievable due to its relatively low incidence, pancreatic cancer surveillance programs are recommended for patients with germline causative variants in PDAC susceptibility genes or a strong family history. In this study, we sought to determine the prevalence and significance of germline alterations in major genes (ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, STK11, TP53) involved in PDAC susceptibility. We performed a systematic review of PubMed publications reporting germline variants identified in these genes in PDAC patients. Overall, the retrieved articles included 1493 PDAC patients. A high proportion of these patients (n = 1225/1493, 82%) were found to harbor alterations in genes (ATM, BRCA1, BRCA2, PALB2) involved in the homologous recombination repair (HRR) pathway. Specifically, the remaining PDAC patients were reported to carry alterations in genes playing a role in other cancer pathways (CDKN2A, STK11, TP53; n = 181/1493, 12.1%) or in the mismatch repair (MMR) pathway (MLH1, MSH2, MSH6, PMS2; n = 87/1493, 5.8%). Our findings highlight the importance of germline genetic characterization in PDAC patients for better personalized targeted therapies, clinical management, and surveillance.
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Colorectal tumorigenesis is driven by alterations in genes and proteins responsible for cancer initiation, progression, and invasion. This multistage process is based on a dense network of protein-protein interactions (PPIs) that become dysregulated as a result of changes in various cell signaling effectors. PPIs in signaling and regulatory networks are known to be mediated by short linear motifs (SLiMs), which are conserved contiguous regions of 3-10 amino acids within interacting protein domains. SLiMs are the minimum sequences required for modulating cellular PPI networks. Thus, several in silico approaches have been developed to predict and analyze SLiM-mediated PPIs. In this review, we focus on emerging evidence supporting a crucial role for SLiMs in driver pathways that are disrupted in colorectal cancer (CRC) tumorigenesis and related PPI network alterations. As a result, SLiMs, along with short peptides, are attracting the interest of researchers to devise small molecules amenable to be used as novel anti-CRC targeted therapies. Overall, the characterization of SLiMs mediating crucial PPIs in CRC may foster the development of more specific combined pharmacological approaches.
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Neoplasias Colorrectales , Proteínas , Humanos , Bases de Datos de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas/metabolismo , Péptidos , Neoplasias Colorrectales/genéticaRESUMEN
c-MYC is one of the most important factors involved in colorectal cancer (CRC) initiation and progression; indeed, it is found to be upregulated in up to 80% of sporadic cases. During colorectal carcinogenesis, c-MYC is maintained upregulated through ß-catenin-mediated transcriptional activation and ERK-mediated post-translational stabilization. Our data demonstrate that p38α, a kinase involved in CRC metabolism and survival, contributes to c-Myc protein stability. Moreover, we show that p38α, like ERK, stabilizes c-MYC protein levels by preventing its ubiquitination. Of note, we found that p38α phosphorylates c-MYC and interacts with it both in vitro and in cellulo. Extensive molecular analyses in the cellular and in vivo models revealed that the p38α kinase inhibitors, SB202190 and ralimetinib, affect c-MYC protein levels. Ralimetinib also exhibited a synthetic lethality effect when used in combination with the MEK1 inhibitor trametinib. Overall, our findings identify p38α as a promising therapeutic target, acting directly on c-MYC, with potential implications for countering c-MYC-mediated CRC proliferation, metastatic dissemination, and chemoresistance.
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SMYD3 overexpression in several human cancers highlights its crucial role in carcinogenesis. Nonetheless, SMYD3 specific activity in cancer development and progression is currently under debate. Taking advantage of a library of rare tripeptides, which we first tested for their in vitro binding affinity to SMYD3 and then used as in silico probes, we recently identified BRCA2, ATM, and CHK2 as direct SMYD3 interactors. To gain insight into novel SMYD3 cancer-related roles, here we performed a comprehensive in silico analysis to cluster all potential SMYD3-interacting proteins identified by screening the human proteome for the previously tested tripeptides, based on their involvement in cancer hallmarks. Remarkably, we identified mTOR, BLM, MET, AMPK, and p130 as new SMYD3 interactors implicated in cancer processes. Further studies are needed to characterize the functional mechanisms underlying these interactions. Still, these findings could be useful to devise novel therapeutic strategies based on the combined inhibition of SMYD3 and its newly identified molecular partners. Of note, our in silico methodology may be useful to search for unidentified interactors of other proteins of interest.
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Genetic variants located in non-coding regions can affect processes that regulate protein expression, functionally contributing to human disease. Germline heterozygous mutations in the non-coding region of the PTEN gene have been previously identified in patients with PTEN hamartoma tumor syndrome (PHTS) diagnosed with breast, thyroid, and/or endometrial cancer. In this study, we report a PTEN promoter variant (rs34149102 A allele) that was identified by direct sequencing in an Italian family with a history of gastroesophageal junction (GEJ) adenocarcinoma and breast cancer. In order to investigate the putative functional role of the rs34149102 A allele variant, we evaluated the status of PTEN alterations at the somatic level. We found that PTEN protein expression was absent in the GEJ adenocarcinoma tissue of the index case. Moreover, we detected the occurrence of copy number loss involving the PTEN rs34149102 major C allele in tumor tissue, revealing that the second allele was somatically inactivated. This variant is located within an active regulatory region of the PTEN core promoter, and in silico analysis suggests that it may affect the binding of the nuclear transcription factor MAZ and hence PTEN expression. Overall, these results reveal the functional role of the PTEN promoter rs34149102 A allele variant in the modulation of PTEN protein expression and highlight its contribution to hereditary cancer risk.
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Adenocarcinoma , Neoplasias de la Mama , Síndrome de Hamartoma Múltiple , Neoplasias de la Mama/genética , Neoplasias Esofágicas , Femenino , Células Germinativas/metabolismo , Síndrome de Hamartoma Múltiple/genética , Humanos , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismoRESUMEN
Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature meta-analysis of APC splicing mutations. We found that 119 unique APC splicing mutations, including the one described here, have been reported in FAP patients, 69 of which have been characterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with four mutations causing skipping of exon 12 or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and five mutations leading to skipping of exon 5, 7, 8, or (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations causing skipping of exon 12 or 13 considered in this study cluster with the AFAP phenotype and reveal a potential molecular mechanism of pathogenesis in FAP disease.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/genética , Exones , Mutación de Línea Germinal , Linaje , Familia , Femenino , Células HEK293 , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The prognosis of locally advanced colorectal cancer (CRC) is currently unsatisfactory. This is mainly due to drug resistance, recurrence, and subsequent metastatic dissemination, which are sustained by the cancer stem cell (CSC) population. The main driver of the CSC gene expression program is Wnt signaling, and previous reports indicate that Wnt3a can activate p38 MAPK. Besides, p38 was shown to feed into the canonical Wnt/ß-catenin pathway. Here we show that patient-derived locally advanced CRC stem cells (CRC-SCs) are characterized by increased expression of p38α and are "addicted" to its kinase activity. Of note, we found that stage III CRC patients with high p38α levels display reduced disease-free and progression-free survival. Extensive molecular analysis in patient-derived CRC-SC tumorspheres and APCMin/+ mice intestinal organoids revealed that p38α acts as a ß-catenin chromatin-associated kinase required for the regulation of a signaling platform involved in tumor proliferation, metastatic dissemination, and chemoresistance in these CRC model systems. In particular, the p38α kinase inhibitor ralimetinib, which has already entered clinical trials, promoted sensitization of patient-derived CRC-SCs to chemotherapeutic agents commonly used for CRC treatment and showed a synthetic lethality effect when used in combination with the MEK1 inhibitor trametinib. Taken together, these results suggest that p38α may be targeted in CSCs to devise new personalized CRC treatment strategies.
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Cromatina/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Células Madre Neoplásicas/metabolismo , Organoides/metabolismo , Procesamiento Proteico-Postraduccional/genética , beta Catenina/metabolismo , Neoplasias Colorrectales/genética , Humanos , PronósticoRESUMEN
SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (KD =42 and 84â µM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3-HSP90 binding was confirmed (KD =13â µM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.