Distinct Driver Pathway Enrichments and a High Prevalence of TSC2 Mutations in Right Colon Cancer in Chile: A Preliminary Comparative Analysis.

Colorectal cancer (CRC) is the second leading cause of cancer deaths globally. While ethnic differences in driver gene mutations have been documented, the South American population remains understudied at the genomic level, despite facing a rising burden of CRC. We analyzed tumors of 40 Chilean CRC patients (Chp) using next-generation sequencing and compared them to data from mainly Caucasian cohorts (TCGA and MSK-IMPACT). We identified 388 mutations in 96 out of 135 genes, with TP53 (45%), KRAS (30%), PIK3CA (22.5%), ATM (20%), and POLE (20%) being the most frequently mutated. TSC2 mutations were associated with right colon cancer (44.44% in RCRC vs. 6.45% in LCRC, p-value = 0.016), and overall frequency was higher compared to TCGA (p-value = 1.847 × 10-5) and MSK-IMPACT cohorts (p-value = 3.062 × 10-2). Limited sample size restricts definitive conclusions, but our data suggest potential differences in driver mutations for Chilean patients, being that the RTK-RAS oncogenic pathway is less affected and the PI3K pathway is more altered in Chp compared to TCGA (45% vs. 25.56%, respectively). The prevalence of actionable pathways and driver mutations can guide therapeutic choices, but can also impact treatment effectiveness. Thus, these findings warrant further investigation in larger Chilean cohorts to confirm these initial observations. Understanding population-specific driver mutations can guide the development of precision medicine programs for CRC patients.

Uncommon Large and Bilateral Fibrous Cephalic Plaques in a Patient with TSC2-Related Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a genetic disorder, frequently characterized by early dermatological manifestations. The recognition and adequate description of these dermatological manifestations are of utmost importance for early diagnosis, allowing for the implementation of therapeutic and preventive measures. Fibrous cephalic plaques (FCPs) are considered a major diagnostic criterion for TSC, as FCPs are the most specific skin lesions of TSC. The localization, consistency, color, and size of FCPs vary widely, which can cause diagnostic delay, especially in patients with atypical presentations. The present report describes a female TSC patient with a confirmed heterozygous pathogenic genotype, NG_005895.1 (TSC2_v001): c.2640-1G>T, who presented with uncommon large and bilateral FCPs causing bilateral ptosis and marked with hyperostosis of the diploe that generated an asymmetry of the brain parenchyma. Differential diagnoses considered initially in this patient due to the atypical FCPs are described.

Clinical, pathological, and molecular correlation of folliculocystic and collagen hamartoma: A new potential diagnostic criterion for tuberous sclerosis complex?

Folliculocystic and collagen hamartoma (FCCH) is a rare entity with only 18 reported cases worldwide. Of them, most are found in patients diagnosed with tuberous sclerosis complex (TSC). FCCH has distinctive histopathologic features, including collagen deposition in the dermis, perifollicular fibrosis, and comedones with keratin-containing cysts lined by infundibular epithelium. We report three patients with a definitive TSC clinical diagnosis in whom clinical, histopathologic, and molecular features were studied to establish if there exists a genotype-phenotype correlation. The molecular results showed different heterozygous pathogenic variants (PV) in TSC2 in each patient: NM_000548.4:c.5024C>T, NG_005895.1:c.1599+1G>T, and NM_000548.4:c.2297_2298dup, to our knowledge; the latter PV has not been reported in public databases. The same PVs were identified as heterozygous in the tumor tissue samples, none of which yielded evidence of a TSC2 second hit. Because all FCCH patients with available molecular diagnosis carry a pathogenic genotype in TSC1 or TSC2, we suggest that FCCH should be considered as a new and uncommon diagnostic manifestation in the TSC consensus international diagnostic criteria. The early recognition of FCCH by clinicians could prompt the identification of new TSC cases. Interestingly, our molecular findings suggest that one of the patients described herein is a probable case of somatic mosaicism.

A novel mutation in the TSC2 gene protects against colorectal cancer in the Mexican population.

INTRODUCTION: Colorectal cancer (CRC) is a complex disease due to the large number of factors that influence its development, including variants in tumor suppressor genes. OBJECTIVE: To estimate allelic and genotypic frequencies of c.3915G>A and c.5371G>A variants of the TSC2 gene in a Mexican population with CRC, as well as to analyze their association with the development of CRC. METHODS: 126 peripheral blood samples from patients diagnosed with sporadic CRC and 134 from healthy individuals, regarded as the control group, were included. Identification of genotypes was carried out using traditional PCR and enzymatic digestion. All individuals signed an informed consent letter. RESULTS: The A allele of the c.3915G>A variant (OR = 0.31, 95% CI = 0.15-0.69, p = 0.004), as well as A/G haplotype of the c.3915G>A and c.5371G>A variants (OR = 0.28, 95% CI = 0.12-0.68, p = 0.005) showed a possible protective effect against sporadic CRC. In silico analysis indicated that both variants generate modifications in the splicing process. CONCLUSION: The presence of TSC2 gene c.3915G>A variant suggests a possible protective effect against sporadic CRC in the Mexican population; however, no association was observed with the c.5371G>A variant.

INTRODUCCIÓN: El cáncer colorrectal (CCR) es una enfermedad compleja debido al gran número de factores que influyen en su desarrollo, incluyendo variantes en genes supresores de tumores. OBJETIVO: Estimar las frecuencias alélicas y genotípicas de las variantes c.3915G>A y c.5371G>A del gen TSC2 en una población mexicana con CCR, así como analizar la asociación con el desarrollo de CCR. MÉTODOS: Se incluyeron 126 muestras de sangre periférica de pacientes con diagnóstico de CCR esporádico y 134 de individuos sanos, considerados como grupo de control. La identificación de los genotipos se llevó a cabo mediante PCR tradicional y digestión enzimática. Todos los individuos firmaron una carta de consentimiento informado. RESULTADOS: El alelo A de la variante c.3915G>A (RM = 0.31, IC 95 % = 0.15-0.69, p = 0.004), así como el haplotipo A/G de las variantes c.3915G>A y c.5371G>A (RM = 0.28, IC 95 % = 0.12-0.68, p = 0.005) mostraron un posible efecto protector contra CCR esporádico. El análisis in silico indicó que ambas variantes generan modificaciones en el proceso de corte y empalme. CONCLUSIÓN: La presencia de la variante c.3915G>A del gen TSC2 sugiere un posible efecto protector contra CCR esporádico en población mexicana; sin embargo, no se observó esta asociación con la variante c.5371G>A.

Nueva mutación en el gen TSC2 protege contra el cáncer colorrectal en población mexicana / A novel mutation in the TSC2 gene protects against colorectal cancer in the Mexican population

Resumen Introducción: El cáncer colorrectal (CCR) es una enfermedad compleja debido al gran número de factores que influyen en su desarrollo, incluyendo variantes en genes supresores de tumores. Objetivo: Estimar las frecuencias alélicas y genotípicas de las variantes c.3915G>A y c.5371G>A del gen TSC2 en una población mexicana con CCR, así como analizar la asociación con el desarrollo de CCR. Métodos: Se incluyeron 126 muestras de sangre periférica de pacientes con diagnóstico de CCR esporádico y 134 de individuos sanos, considerados como grupo de control. La identificación de los genotipos se llevó a cabo mediante PCR tradicional y digestión enzimática. Todos los individuos firmaron una carta de consentimiento informado. Resultados: El alelo A de la variante c.3915G>A (RM = 0.31, IC 95 % = 0.15-0.69, p = 0.004), así como el haplotipo A/G de las variantes c.3915G>A y c.5371G>A (RM = 0.28, IC 95 % = 0.12-0.68, p = 0.005) mostraron un posible efecto protector contra CCR esporádico. El análisis in silico indicó que ambas variantes generan modificaciones en el proceso de corte y empalme. Conclusión: La presencia de la variante c.3915G>A del gen TSC2 sugiere un posible efecto protector contra CCR esporádico en población mexicana; sin embargo, no se observó esta asociación con la variante c.5371G>A.

Abstract Introduction: Colorectal cancer (CRC) is a complex disease due to the large number of factors that influence its development, including variants in tumor suppressor genes. Objective: To estimate allelic and genotypic frequencies of c.3915G>A and c.5371G>A variants of the TSC2 gene in a Mexican population with CRC, as well as to analyze their association with the development of CRC. Methods: 126 peripheral blood samples from patients diagnosed with sporadic CRC and 134 from healthy individuals, regarded as the control group, were included. Identification of genotypes was carried out using traditional PCR and enzymatic digestion. All individuals signed an informed consent letter. Results: The A allele of the c.3915G>A variant (OR = 0.31, 95% CI = 0.15-0.69, p = 0.004), as well as A/G haplotype of the c.3915G>A and c.5371G>A variants (OR = 0.28, 95% CI = 0.12-0.68, p = 0.005) showed a possible protective effect against sporadic CRC. In silico analysis indicated that both variants generate modifications in the splicing process. Conclusion: The presence of TSC2 gene c.3915G>A variant suggests a possible protective effect against sporadic CRC in the Mexican population; however, no association was observed with the c.5371G>A variant.

Blocking the Farnesyl Pocket of PDEδ Reduces Rheb-Dependent mTORC1 Activation and Survival of Tsc2-Null Cells.

Rheb is a small GTPase member of the Ras superfamily and an activator of mTORC1, a protein complex master regulator of cell metabolism, growth, and proliferation. Rheb/mTORC1 pathway is hyperactivated in proliferative diseases, such as Tuberous Sclerosis Complex syndrome and cancer. Therefore, targeting Rheb-dependent signaling is a rational strategy for developing new drug therapies. Rheb activates mTORC1 in the cytosolic surface of lysosomal membranes. Rheb's farnesylation allows its anchorage on membranes, while its proper localization depends on the prenyl-binding chaperone PDEδ. Recently, the use of PDEδ inhibitors has been proposed as anticancer agents because they interrupted KRas signaling leading to antiproliferative effects in KRas-dependent pancreatic cancer cells. However, the effect of PDEδ inhibition on the Rheb/mTORC1 pathway has been poorly investigated. Here, we evaluated the impact of a new PDEδ inhibitor, called Deltasonamide 1, in Tsc2-null MEFs, a Rheb-dependent overactivated mTORC1 cell line. By using a yeast two-hybrid assay, we first validated that Deltasonamide 1 disrupts Rheb-PDEδ interaction. Accordingly, we found that Deltasonamide 1 reduces mTORC1 targets activation. In addition, our results showed that Deltasonamide 1 has antiproliferative and cytotoxic effects on Tsc2-null MEFs but has less effect on Tsc2-wild type MEFs viability. This work proposes the pharmacological PDEδ inhibition as a new approach to target the abnormal Rheb/mTORC1 activation in Tuberous Sclerosis Complex cells.

Long non-coding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy inhibition by regulating TSC2-mTOR signaling.

BACKGROUND: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. METHODS: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. RESULTS: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. CONCLUSION: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.

Long non-coding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy inhibition by regulating TSC2-mTOR signaling

BACKGROUND: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. METHODS: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. RESULTS: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. CONCLUSION: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.

Calcitriol Reverses the Down-Regulation Pattern of Tuberous Sclerosis Complex Genes in an In Vitro Calcification Model.

Tuberous sclerosis complex (TSC) is a neurocutaneous syndrome with autosomal dominant inheritance, and most of the cases are related to loss of function of the TSC1 and TSC2 genes. TSC may occur with a wide range of clinical findings and skin, kidney, brain, and heart are the most commonly affected organs. Brain calcifications in TSC are also described and reported as diffuse and without pattern of symmetry or bilaterality. Recently, a new discovery opened the possibility of using vitamin D (VitD) for treating cerebral calcifications. Calcitriol, the active form of VitD, was able to reduce the calcification in an in vitro model, increasing expression of a gene related to primary familial brain calcification. We show that in the same experimental model, calcitriol was also able to restore and even increase expression of genes related to TSC. This article discusses the use of calcitriol supplementation in patients with TSC, which can be a very interesting strategy due to its low cost and because it is already used in various therapies.

TSC1 and TSC2 gene mutations and their implications for treatment in Tuberous Sclerosis Complex: a review

Abstract Tuberous sclerosis complex is an autosomal dominant disorder characterized by skin manifestations and formation of multiple tumors in different organs, mainly in the central nervous system. Tuberous sclerosis is caused by the mutation of one of two tumor suppressor genes, TSC1 or TSC2. Currently, the development of novel techniques and great advances in high-throughput genetic analysis made mutation screening of the TSC1 and TSC2 genes more widely available. Extensive studies of the TSC1 and TSC2 genes in patients with TSC worldwide have revealed a wide spectrum of mutations. Consequently, the discovery of the underlying genetic defects in TSC has furthered our understanding of this complex genetic disorder, and genotype-phenotype correlations are becoming possible, although there are still only a few clearly established correlations. This review focuses on the main symptoms and genetic alterations described in TSC patients from 13 countries in three continents, as well as on genotype-phenotype correlations established to date. The determination of genotype-phenotype correlations may contribute to the establishment of successful personalized treatment for TSC.

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration.

Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.