Dissection moléculaire de la sénescence cellulaire induite par le stress et la thérapie dans le cancer de l’ovaire et son impact sur la réponse des patientes

Le cancer de l’ovaire (COv) est le cancer gynécologique le plus létal chez la femme et les traitements existants, chirurgie et chimiothérapie, ont peu évolué au cours des dernières décennies. Nous proposons que la compréhension des différents destins cellulaires tels que la sénescence que peuvent choisir les cellules du cancer de l’ovaire en réponse à la chimiothérapie pourrait conduire à de nouvelles opportunités thérapeutiques. La sénescence cellulaire a été largement associée à l’activité de la protéine TP53, qui est mutée dans plus de 90% des cas de cancer de l’ovaire séreux de haut grade (COv-SHG), la forme la plus commune de la maladie. Dans nos travaux, à partir d’échantillons dérivés de patientes, nous montrons que les cultures primaires du cancer de l’ovaire séreux de haut grade exposées au stress ou à des drogues utilisées en chimiothérapie entrent en senescence grâce à l’activité d’un isoforme du gène CDKN2A (p16INK4A). Dans ces cellules, nous avons évalué les caractéristiques fondamentales de la sénescence cellulaire tels que les altérations morphologiques, l’activité béta galactosidase associée à la sénescence, les dommages à l’ADN, l’arrêt du cycle cellulaire et le phénotype sécrétoire associé à la sénescence. En utilisant des micromatrices tissulaires construites à partir d’échantillons humains de COv-SHG pré- et post-chimiothérapie, accompagnées de leurs données cliniques, nous avons quantifié des marqueurs de sénescence incluant une diminution de la prolifération cellulaire quelques semaines après chimiothérapie. De façon intéressante, l’expression de p16INK4A dans les échantillons de COv-SHG prétraitement corrèle avec une survie prolongée des patientes suite au traitement. Ceci suggère ainsi pour la première fois un impact biologique bénéfique pour la présence de cellules cancéreuses qui sont capable d’activer la sénescence, particulièrement pour le traitement du cancer de l’ovaire. Dans le but de complémenter les thérapies actuelles avec des approches de manipulation pharmacologique de la sénescence, nos résultats suggèrent qu’il serait important de déterminer l’impact positif ou négatif de la sénescence induite par la thérapie sur la progression de la maladie et la survie, pour chaque type de cancer de façon indépendante.

Allelic imbalance studies of chromosome 9 suggest major differences in chromosomal instability among nonmelanoma skin carcinomas

CONTEXTO: Vários estudos de perda de heterozigozidade na região 9p21-p22, que abriga os genes supressores tumorais CDKN2a/p16INK4a, p19ARF e p15INK4b, têm sido realizados em uma ampla série de tumores humanos, incluindo os melanomas familiares. Perdas e ganhos em outras regiões do cromossomo 9 também têm sido observados com freqüência e podem indicar mecanismos adicionais no processo de tumorigênese dos carcinomas basocelulares da pele. OBJETIVO: Investigar o equilíbrio alélico existente na região 9p21-p22 em carcinomas basocelulares. TIPO DE ESTUDO: Análise molecular de marcadores de microssatélites em tumores e controles. LOCAL: Dois serviços de dermatologia de atendimento terciário em universidades públicas de São Paulo e o Laboratório de Genética Molecular do Câncer da Universidade Estadual de Campinas (UNICAMP), Brasil. PARTICIPANTES: Examinamos 13 casos benignos, incluindo 4 queratoses solares, 3 queratoacantomas, 3 nevos melanocíticos, 2 doenças de Bowen e 1 neurofibroma cutâneo, além de 58 tumores malignos da pele: 14 de células escamosas, 40 carcinomas basocelulares e 4 melanomas; em pacientes consecutivamente encaminhados à clínica de Dermatologia da Unicamp e que concordaram em participar do estudo. VARIÁVEIS ESTUDADAS: O tumor principal e uma porção normal de pele não-adjacente foram removidos cirurgicamente de pacientes que consecutivamente procuraram os ambulatórios de dermatologia da Universidade Estadual de Campinas (UNICAMP) e da Universidade Estadual de São Paulo (Unesp), São Paulo, por causa de lesões cutâneas. Extraímos DNA tanto de tecido tumoral como do correspondente tecido normal de cada paciente. Para amplificar regiões de repetição polimórfica de microssatélites do cromossomo 9, foram utilizados quatro pares de primers, sendo dois deles destinados à região 9p21-p22. RESULTADOS: Identificamos oito casos (20%) de desequilíbrio alélico entre os carcinomas basocelulares, sendo dois casos de perda de heterozigozidade e seis casos de instabilidade de microssatélite na região 9p21-p22. Outros marcadores também mostravam anormalidades em três destes tumores, enquanto nenhuma alteração foi detectada entre os casos benignos e nos outros tumores malignos. CONCLUSÃO: Esta dependência fenotípica sugere que existem diferenças importantes no comportamento das formas mais comuns de tumores cutâneos não-melanocíticos em relação à sua tendência para instabilidade de microssatélite no cromossomo 9. Considerando-se que os genes CDKN2a/p16INK4a, p19ARF e p15INK4b não parecem responsáveis pelas anormalidades observadas, outros genes em 9p21-p22 podem estar envolvidos na etiopatogenia e na progressão dos carcinomas basocelulares.

CDKN2A promoter methylation is related to the tumor location and histological subtype and associated with Helicobacter pylori flaA(+) strains in gastric adenocarcinomas

Promoter hypermethylation of CDKN2A (p16INK4A protein) is the main mechanism of gene inactivation. However, its association with Helicobacter pylori infection is a controversial issue. Therefore, we examined a series of gastric adenocarcinomas to assess the association between p16INK4A inactivation and H. pylori genotype (vacA, cagA, cagE, virB11 and flaA) according to the location and histological subtype of the tumors. p16INK4A expression and CDKN2A promoter methylation were found in 77 gastric adenocarcinoma samples by immunohistochemistry and methylation-specific PCR, respectively. Helicobacter pylori infection and genotype were determined by PCR. A strong negative correlation between immunostaining and CDKN2A promoter region methylation was found. In diffuse subtype tumors, the inactivation of p16INK4A by promoter methylation was unique in noncardia tumors (p = 0.022). In addition, H. pylori-bearing flaA was associated with non-methylation tumors (p = 0.008) and H. pylori strain bearing cagA or vacAs1m1 genes but without flaA was associated with methylated tumors (p = 0.022 and 0.003, respectively). Inactivation of p16INK4A in intestinal and diffuse subtypes showed distinct carcinogenic pathways, depending on the tumor location. Moreover, the process of methylation of the CDKN2A promoter seems to depend on the H. pylori genotype. The present data suggest that there is a differential influence and relevance of H. pylori genotype in gastric cancer development.

CDKN2A promoter hypermethylation in astrocytomas is associated with age and sex

CDKN2A promoter hypermethylation has been widely related to many cancers. In astrocytomas, although CDKN2A (p16INK4A protein) is often inactivated, there are still some controversial issues regarding the mechanism by which this alteration occurs. Thus, we analyzed a series of astrocytomas to assess the association between CDKN2A expression and methylation of grade I-IV tumors (WHO) and clinicopathological parameters. DNA extracted from formalin-fixed paraffin-embedded material of 93 astrocytic tumors was available for CDKN2A promoter methylation analysis and p16INK4A expression by methylation-specific PCR and immunohistochemistry, respectively. A strong negative correlation between nuclear and cytoplasmic immunostaining and CDKN2A promoter methylation was found. Additionally, a significant negative correlation between CDKN2A promoter methylation and age was observed; also, female patients had statistically more CDKN2A methylated promoters (p=0.036) than men. In conclusion, CDKN2A inactivation by promoter methylation is a frequent event in astrocytomas and it is related to the age and sex of patients. © 2013 Surgical Associates Ltd.

Genotipagem de HPV e imunorreatividade da ciclina D1 em adenocarcinoma de colo do útero

Pós-graduação em Patologia - FMB

The regulation of Satellite Cells during skeletal muscle regeneration and neuromuscular disease.

Skeletal muscle possesses the remarkable capacity to complete a rapid and extensive regeneration, even following severe damage. The regenerative ability of skeletal muscle relies on Satellite Cells (SCs), a population of muscle specific adult stem cells. However, during aging or under several pathological conditions, the ability of skeletal muscle to fully regenerated is compromised. Here, a morphological and molecular study on SCs from patients affected by ALS is described. Moreover, the role of the cell cycle regulator P16Ink4a during skeletal muscle regeneration and aging has been investigated.

Profili di espressione genetica ed epigenetica ad impatto prognostico e predittivo nel carcinoma squamoso e nelle lesioni potenzialmente maligne del cavo orale

Il carcinoma squamoso orale (CSO) è spesso preceduto da lesioni definite potenzialmente maligne tra cui la leucoplachia e il lichen ma una diagnosi precoce avviene ancora oggi in meno della metà dei casi. Inoltre spesso un paziente trattato per CSO svilupperà secondi tumori. Scopo del lavoro di ricerca è stato: 1) Studiare, mediante metodica di next generation sequencing, lo stato di metilazione di un gruppo di geni a partire da prelievi brushing del cavo orale al fine di identificare CSO o lesioni ad alto rischio di trasformazione maligna. 2) Valurare la relazione esistente tra sovraespressione di p16INK4A e presenza di HPV in 35 pazienti affetti da lichen 3) Valutare la presenza di marker istopatologici predittivi di comparsa di seconde manifestazioni tumorali 4) valutare la relazione clonale tra tumore primitivo e metastasi linfonodale in 8 pazienti mediante 2 metodiche di clonalità differenti: l’analisi di mtDNA e delle mutazioni del gene TP53. I risultati hanno mostrato: 1) i geni ZAP70 e GP1BB hanno presentato un alterato stato di metilazione rispettivamente nel 100% e nel 90,9% di CSO e lesioni ad alto rischio, mentre non sono risultati metilati nei controlli sani; ipotizzando un ruolo come potenziali marcatori per la diagnosi precoce nel CSO. 2)Una sovraespressione di p16INK4A è risultata in 26/35 pazienti affetti da lichen ma HPV-DNA è stato identificato in soli 4 campioni. Nessuna relazione sembra essere tra sovraespressione di p16INK4A e virus HPV. 3)L’invasione perineurale è risultato un marker predittivo della comparsa di recidiva locale e metastasi linfonodale, mentre lo stato dei margini chirurgici si è rilevato un fattore predittivo per la comparsa di secondi tumori primitivi 4) Un totale accordo nei risultati c’è stato tra analisi di mtDNA e analisi di TP53 e le due metodiche hanno identificato la presenza di 4 metastasi linfonodali non clonalmente correlate al tumore primitivo.

Stratification and Prognostic Relevance of Jass's Molecular Classification of Colorectal Cancer

Background: The current proposed model of colorectal tumorigenesis is based primarily on CpG island methylator phenotype (CIMP), microsatellite instability (MSI), KRAS, BRAF, and methylation status of 0-6-Methylguanine DNA Methyltransferase (MGMT) and classifies tumors into five subgroups. The aim of this study is to validate this molecular classification and test its prognostic relevance. Methods: Three hundred two patients were included in this study. Molecular analysis was performed for five CIMP-related promoters (CRABP1, MLH1, p16INK4a, CACNA1G, NEUROG1), MGMT, MSI, KRAS, and BRAF. Methylation in at least 4 promoters or in one to three promoters was considered CIMP-high and CIMP-low (CIMP-H/L), respectively. Results: CIMP-H, CIMP-L, and CIMP-negative were found in 7.1, 43, and 49.9% cases, respectively. One hundred twenty-three tumors (41%) could not be classified into any one of the proposed molecular subgroups, including 107 CIMP-L, 14 CIMP-H, and two CIMP-negative cases. The 10 year survival rate for CIMP-high patients [22.6% (95%CI: 7-43)] was significantly lower than for CIMP-L or CIMP-negative (p = 0.0295). Only the combined analysis of BRAF and CIMP (negative versus L/H) led to distinct prognostic subgroups. Conclusion: Although CIMP status has an effect on outcome, our results underline the need for standardized definitions of low- and high-level CIMP, which clearly hinders an effective prognostic and molecular classification of colorectal cancer.

Limited redundancy in phosphorylation of retinoblastoma tumor suppressor protein by cyclin-dependent kinases in acute lymphoblastic leukemia

Cyclin-dependent kinases (CDKs) successively phosphorylate the retinoblastoma protein (RB) at the restriction point in G1 phase. Hyperphosphorylation results in functional inactivation of RB, activation of the E2F transcriptional program, and entry of cells into S phase. RB unphosphorylated at serine 608 has growth suppressive activity. Phosphorylation of serines 608/612 inhibits binding of E2F-1 to RB. In Nalm-6 acute lymphoblastic leukemia extracts, serine 608 is phosphorylated by CDK4/6 complexes but not by CDK2. We reasoned that phosphorylation of serines 608/612 by redundant CDKs could accelerate phospho group formation and determined which G1 CDK contributes to serine 612 phosphorylation. Here, we report that CDK4 complexes from Nalm-6 extracts phosphorylated in vitro the CDK2-preferred serine 612, which was inhibited by p16INK4a, and fascaplysin. In contrast, serine 780 and serine 795 were efficiently phosphorylated by CDK4 but not by CDK2. The data suggest that the redundancy in phosphorylation of RB by CDK2 and CDK4 in Nalm-6 extracts is limited. Serine 612 phosphorylation by CDK4 also occurred in extracts of childhood acute lymphoblastic leukemia cells but not in extracts of mobilized CD34+ hemopoietic progenitor cells. This phenomenon could contribute to the commitment of childhood acute lymphocytic leukemia cells to proliferate and explain their refractoriness to differentiation-inducing agents.

E2F1 AND TUMOR SUPPRESSION: THE ROLE OF p21, miRNAS, AND THE DNA DAMAGE RESPONSE

E2F1 is a multi-faceted protein that has roles in a number of important cellular processes including cell cycle regulation, apoptosis, proliferation, and the DNA damage response (DDR). Moreover, E2F1 has opposing roles in tumor development, acting as either a tumor suppressor or an oncogene depending on the context. In human cancer, E2F1 is often deregulated through aberrations in the Rb-p16INK4a-cyclin D1 pathway. In these studies we examined three mechanisms by which E2F1 might mediate its tumor suppressive properties: p21-induced senescence, miRNAs, and the DNA damage response. We found that E2F1 acts as a tumor suppressor in response to ras activation through a non-apoptotic mechanism requiring ARF and p53, but not p21. However, p21-loss inhibited two-stage chemical carcinogenesis in FVB mice. In response to E2F1 overexpression, we found that 22 miRNAs are differentially regulated in mouse epidermis, including let-7a, let-7c, and miR-301. Additionally, regulation of miR-301 involves binding of E2F1 to its promoter. Finally, our data indicate a role for E2F1 at sites of DNA damage requiring E2F1’s phosphorylation at serine 31 which may involve DNA repair. Further, this role in the DDR may affect tumor aggressiveness and multiplicity. In all, we have explored three mechanisms for E2F1-induced tumor suppression and identified E2F1’s role in the DNA damage response as a likely contributor to this phenomenon.

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21cip1. and p16INK4a, and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15INK4b by transforming growth factor β treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15INK4b-independent transforming growth factor β-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16INK4a protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.

p19Arf induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation

The Ink4a/Arf locus encodes p16Ink4a and p19Arf and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19Arf and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a/Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16Ink4a and p19Arf are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a/Arf null mice revealed that these cells bypassed crisis. Because expression of p19Arf but not p16 Ink4a induced apoptosis in Ab-MLV-transformed pre-B cells, p19Arf appears to be responsible for these events. Consistent with the link between p19Arf and p53, Ink4a/Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19Arf is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19Arf–p53 regulatory loop plays an important role in lymphoma induction.

Human and Yeast Cdk-activating Kinases (CAKs) Display Distinct Substrate Specificities

Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40MO15/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40MO15 have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40MO15 preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40MO15 only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21CIP1, p27KIP1, p57KIP2, p16INK4a, and p18INK4c, could block phosphorylation by p40MO15 but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40MO15 activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.

The cyclin-dependent kinase inhibitor p27Kip1 induces N-terminal proteolytic cleavage of cyclin A

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27Kip1 (p27) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G1. Here we report that p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs (p15Ink4b, p16Ink4a, p21Cip1, and p57Kip2) do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. We refer to this protease as Tsap (pTwenty-seven- activated protease).