RESUMEN
Alport syndrome (AS) is a rare genetic disorder categorized by the progressive loss of kidney function, sensorineural hearing loss and eye abnormalities. It occurs due to mutations in three genes that encode for the alpha chains of type IV collagen. Globally, the disease is classified based on the pattern of inheritance into X-linked AS (XLAS), which is caused by pathogenic variants in COL4A5, representing 80% of AS. Autosomal recessive AS (ARAS), caused by mutations in either COL4A3 or COL4A4, represents 15% of AS. Autosomal dominant AS (ADAS) is rare and has been recorded in 5% of all cases due to mutations in COL4A3 or COL4A4. This review provides updated knowledge about AS including its clinical and genetic characteristics in addition to available therapies that only slow the progression of the disease. It also focuses on reported cases in Saudi Arabia and their prevalence. Moreover, we shed light on advances in genetic technologies like gene editing using CRISPR/Cas9 technology, the need for an early diagnosis of AS and managing the progression of the disease. Eventually, we provide a few recommendations for disease management, particularly in regions like Saudi Arabia where consanguineous marriages increase the risk.
RESUMEN
OBJECTIVES: Although human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients typically experience excellent survival, 15-20 % of patients recur after treatment with chemotherapy and radiation. Therefore, there is a need for biomarkers of treatment failure to guide treatment intensity. MATERIALS AND METHODS: Whole genome sequencing was carried out on HPV+OPSCC patients who were primarily treated with concurrent chemotherapy (cisplatin) and radiation. We then explored whether the loss of LRP1Bwas sufficient to drive an aggressive phenotype, and promote a resistance to cisplatin and radiation therapy both in vitro using HPV+ cell lines (93VU147T, UMSCC47, UWO37 and UWO23) and in vivo. RESULTS: Through integrative genomic analysis of three HPV+OPSCC tumour datasets, we identified that deletion of LRP1B was enriched in samples that recurred following chemo-radiation. Knockdown using siRNA in four HPV+ cell lines (UWO23, UWO37, UMSCC47 and 93VU147T) resulted in increased proliferation of all cases. CRISPR/Cas9 deletion of LRP1B in the same cell line panel demonstrated increased proliferation, clonogenic growth and migration, as well as resistance to both cisplatin and radiation in LRP1B deleted cells compared to their respective non-targeting control cells. Cell line derived xenograft studies indicated that the LRP1B knockout tumours were more resistant to cisplatin and radiation therapy compared to their controls invivo. CONCLUSION: Taken together, our work implicates LRP1B deletion as a potential biomarker for identifying treatment resistant HPV+ OPSCC cases.
Asunto(s)
Carcinoma de Células Escamosas , Resistencia a Antineoplásicos , Neoplasias de Cabeza y Cuello , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Tolerancia a Radiación , Humanos , Carcinoma de Células Escamosas/patología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/radioterapia , Recurrencia Local de Neoplasia , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Receptores de LDL/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapiaRESUMEN
INTRODUCTION: One of the noted areas of weakness for internal medicine residents is pharmacology. However, there is little data documenting the creation and effect of a comprehensive pharmacology didactic program. Our goal was to create a two-year clinical pharmacology didactic program focused on areas of stated weakness and to evaluate this program for an increase in knowledge and prescribing confidence of the participants. METHODS: From August 2020 to June 2022, a two-year pharmacology program was developed, which included 20 didactic lectures on a variety of topics. Pre- and post-tests were given for 15 of the lectures, and four surveys were given, two during each year of the program. Four questions on each survey were the same and asked about confidence in choosing an appropriate medication based on current guidelines, patient-specific factors, primary literature, and pharmacokinetics. RESULTS: Over the two years, participation in the pharmacology sessions ranged from 17 to 29 residents (65-74% of the residency class). The average pre- and post-test scores increased by an average of 25.1%, which was a statistically significant increase (p<0.001, 95% CI [17.5, 32.8]). A Kruskal-Wallis H test showed a statistically significant difference in resident-reported confidence adjusting medications based on primary literature between the different survey groups, χ2 = 9.871, p = 0.02. CONCLUSION: A two-year, didactic pharmacology program improved the knowledge of resident participants and confidence in their ability to choose an appropriate medication based on primary literature.
RESUMEN
BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.
Asunto(s)
Neoplasias de Cabeza y Cuello , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Humanos , Infecciones por Papillomavirus/complicaciones , Estudios Retrospectivos , Recurrencia Local de Neoplasia , Neoplasias Orofaríngeas/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello , Pronóstico , Biomarcadores , Virus del Papiloma Humano , PapillomaviridaeRESUMEN
BACKGROUND: Pilot and feasibility studies (PAFS) are smaller investigations seeking to assess the feasibility of conducting a larger more definitive study. In late 2016, the CONSORT statement was extended to disseminate good practices for reporting of randomized pilot and feasibility trials. In this quality assurance review, we assessed whether PAFS in the top dental speciality journals adhere to good practices of conduct and reporting, by prioritizing assessment of feasibility and stating pre-defined progression criteria to inform the decision to pursue funding for a larger trial. METHODS: With the help of a librarian, we searched MEDLINE and EMBASE from 2017 to 2020, inclusive, for PAFS in the top 3 journals from each of the 10 dental specialties. We collected data on methodological and general characteristics of the studies, their objectives, and reporting of items recommended in the CONSORT extension. RESULTS: Of the 111 trials included, 51.4% (95% CI 41.7-61.0%) stated some indication of intent to assess feasibility while zero reported progression criteria; 74.8% (95% CI 65.6-82.5%) of trials used the terms "pilot" or "feasibility" in their titles and 82.9% (95% CI 74.6-89.4%) of studies stated there is a need for a future trial, but only 9.0% (95% CI 4.4-15.9%) stated intent to proceed to one. Most of the studies, 53.2% (95% CI 43.4-62.7%), reported hypothesis testing without cautioning readers on the generalizability of the results. Studies that used the terms "pilot" or "feasibility" in their title were less likely to have feasibility objectives, compared to trials that did not, with an odds ratio (OR) of 0.310 (95% CI 0.103-0.930; p = 0.037). Compared to trials that did not conduct hypothesis testing, trials that conducted hypothesis testing were significantly less likely to assess feasibility, among them, trials that cautioned readers on the generalizability of their results had an OR of 0.038 (95% CI 0.005-0.264; p < 0.001) and trials that did not caution readers on the generalizability of their results had an OR of 0.043 (95% CI 0.008-0.238; p = 0.001). CONCLUSION: Many PAFS in dentistry are not conducted with the intent of assessing feasibility, nor do they state progression criteria, and few report intent to proceed to a future trial. Misconceptions about PAFS can lead to them being poorly conducted and reported, which has economic and ethical implications. Research ethics boards, funding agencies, and journals need to raise their standards for the conduct and reporting of PAFS, and resources should be developed to address misconceptions and help guide researchers on the best practices for their conduct and reporting.
RESUMEN
BACKGROUND: Suicidal behaviour remains a major public health concern and countries have responded by authoring guidelines to help mitigate death by suicide. Guidelines can include family-based recommendations, but evidence for the level and category of family-based involvement that is needed to effectively prevent suicide is unclear. AIMS: To explore the association between family-based recommendations in guidelines and countries' crude suicide rates. PROSPERO registration: CRD42019130195. METHOD: MEDLINE, Embase, PsycInfo, Web of Science and WHO MiNDbank databases and grey literature were searched within the past 20 years (1 January 2000 to 22 June 2020) for national guidelines giving family-based recommendations in any of three categories (prevention, intervention and postvention). RESULTS: We included 63 guidelines from 46 countries. All identified guidelines included at least one family-based recommendation. There were no statistically significant differences seen between mean World Health Organization crude suicide rates for countries that included only one, two or all three categories of family-based recommendations. However, a lower spread of crude suicide rates was seen when guideline recommendations included all three categories (mean crude suicide rates for one category: 11.09 (s.d. = 5.71); for two categories: 13.42 (s.d. = 7.76); for three categories: 10.68 (s.d. = 5.20); P = 0.478). CONCLUSIONS: Countries should work towards a comprehensive national suicide guideline that includes all categories of family-based recommendations. Countries with previously established guidelines should work towards the inclusion of evidence-based recommendations that have clear implementation plans to potentially help lower suicide rates.
RESUMEN
BACKGROUND: Numerous studies of head and neck squamous cell carcinoma (HNSCC) have demonstrated disparate outcomes by race and ethnicity. Beyond known associations with socioeconomic variables, whether these are also associated with differences in tumor molecular composition has thus far been poorly explored. METHODS: We downloaded clinical and multiplatform molecular data from The Cancer Genome Atlas and other published studies. These were compared between non-Hispanic Black (n = 43) and White (n = 354) patients with non-HPV-related tumors, using multivariable models. Publicly available validation cohorts were used. RESULTS: Black patients had poorer progression-free survival than White patients. Tumors of Black patients had greater copy number aberrations, and increased SFRP1 methylation and miRNA-mediated PRG4 silencing associated with poor survival. PI3K/AkT/mTOR pathway proteins were differentially expressed. CONCLUSIONS: There are molecular differences between tumors of Black and White patients that may partially account for differences in survival. These may inform targeted treatment decisions to achieve equitable outcomes.
Asunto(s)
Población Negra , Neoplasias de Cabeza y Cuello , Disparidades en el Estado de Salud , Carcinoma de Células Escamosas de Cabeza y Cuello , Neoplasias de Cabeza y Cuello/etnología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/etnología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Tasa de Supervivencia , Población Blanca/genéticaRESUMEN
BACKGROUND: Pilot and feasibility studies (PAFS) often have complex objectives aimed at assessing feasibility of conducting a larger study. These may not be clear to participants in pilot studies. METHODS: Here, we aimed to assess the transparency of informed consent in PAFS by investigating whether researchers communicate, through patient information leaflets and consent forms, key features of the studies. We collected this data from original versions of these documents submitted for ethics approval and the final approved documents for PAFS submitted to the Hamilton Integrated Research Ethics Board, Canada. RESULTS: One hundred eighty-four PAFS, submitted for ethics approval from 2004 to 2020, were included, and we found that of the approved consent documents which were provided to participants, 83.2% (153) stated the terms "pilot" or "feasibility" in their title, 12% (22) stated the definition of a pilot/feasibility study, 42.4% (78) of the studies stated their intent to assess feasibility, 19.6% (36) stated the specific feasibility objectives, 1.6% (3) stated the criteria for success of the pilot study, and 0.5% (1) stated all five of these criteria. After ethics review, a small increase in transparency occurred, ranging from 1.6 to 2.8% depending on the criteria. By extracting data from the protocols of the PAFS, we found that 73.9% (136) stated intent to assess feasibility, 71.2% (131) stated specific feasibility objectives, and 33.7% (62) stated criteria for success of the study to lead to a larger study. CONCLUSION: The transparency of informed consent in PAFS is inadequate and needs to be specifically addressed by research ethics guidelines. Research ethics boards and researchers ought to be made aware and mindful of best practices of informed consent in the context of PAFS.
RESUMEN
OBJECTIVE: Single-photon emission computed tomography (SPECT) is an ancillary noninvasive test commonly used to identify the epileptogenic zone. However, its proper utilization may be limited depending on the resources available at each center. This study aimed to investigate the utility of SPECT in presurgical evaluation of children at our center. METHODS: This was an observational retrospective study in 150 children who were admitted to the Epilepsy Monitoring Unit for presurgical evaluation between 2012 and 2019. The utility of interictal and ictal SPECT in different clinical cohorts was analyzed. RESULTS: Only interictal SPECT was performed on 87 patients (58 %), while ictal SPECT was performed on 62 (41.3 %), and interictal SPECT alone was considered of low diagnostic value. Ictal SPECT was unremarkable in 27 of 62 patients and abnormal in 35. Ictal SPECT was localized to the temporal lobe in 62.8 % and to the extratemporal lobe in 25.7 % of the patients and lateralized to one hemisphere in 11.4 % of the patients. In the abnormal SPECT group, ictal SPECT was considered unnecessary in 7/35 (20 %) patients with a single lesion and 3/35 (8.5 %) patients with a hemispheric lesion. In the remaining 25 patients, surgery was recommended more frequently than invasive EEG monitoring (IEM) for diffuse lesion cases (P = 0.03), while IEM was recommended more frequently than surgery for MRI-negative cases (P = 0.03), and in this group none of the MRI-negative patients underwent surgery. In our entire cohort, epilepsy surgery was performed on 24.4 % of the patients, 64 % with a single lesion, 7.6 % with a hemispheric lesion, 25.6 % with a diffuse lesion, and only one MRI-negative patient (2.5 %). Surgery was performed in 48.7 % of single lesion cases and 20.5 % of diffuse lesion cases with either unremarkable or no ictal SPECT. Engel class I outcome was achieved in 62 % and class II outcome in 33 % of the patients. In the single lesion etiology, 72 % (18/25) patients achieved excellent outcome and within this group, 22 % (4/18) patients had a positive ictal SPECT whereas 78 % (14/18) patients either did not get an ictal SPECT or it was unremarkable. SIGNIFICANCE: Based on our findings, we suggest carefully selecting patients for SPECT imaging to improve its utility and prevent overutilization and potential harm to children.
Asunto(s)
Epilepsia del Lóbulo Temporal/cirugía , Epilepsia/cirugía , Lóbulo Temporal/cirugía , Tomografía Computarizada de Emisión de Fotón Único , Adolescente , Adulto , Niño , Electroencefalografía/métodos , Femenino , Humanos , Masculino , Estudios Retrospectivos , Lóbulo Temporal/patología , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is characterized by aggressive behavior with a tendency for recurrence and metastasis. Analyses of The Cancer Genome Atlas (TCGA) data and other cohort studies suggest that the loss of the chromosomal 3p arm is a frequent genetic event observed in both human papillomavirus positive and negative HNSCC. Early molecular analyses (i.e. RFLP, CGH) identified three common regions (3p14.2, 3p21.3 and 3p25) that frequently exhibited loss of genetic material on one arm of the 3p chromosome. More recently, next generation sequencing has revealed the loss of larger regions of this arm. Here we review the role of chromosomal 3p arm loss in early initiation and progression of HNSCC, and its relationship with poor patient prognosis.
RESUMEN
INTRODUCTION: Suicidal behaviour remains a major public health challenge worldwide. Several countries have developed national suicide guidelines aimed at raising awareness of and preventing deaths by suicide. One of the interventions often mentioned in these national guidelines is the involvement of family members as a protective factor in suicide prevention. However, the level or type of family involvement required to reduce suicidal behaviour is not well understood. Thus, in this systematic review, we seek to determine the effectiveness of family-based interventions as a suicide prevention tool, by comparing suicide mortality rates between countries whose national suicide prevention guidelines include family-based interventions and those whose do not. METHODS AND ANALYSIS: MEDLINE, EMBASE, PsycINFO, Web of Science and WHO MiNDbank databases as well as grey literature such as National Guideline Clearinghouse will be searched. National guidelines for suicide prevention published within the last 20 years (between 1999 and 2019) will be included. Results will be analysed using thematic and qualitative analyses. ETHICS AND DISSEMINATION: The findings of the study will help improve the efficacy of national suicide prevention strategies. Findings will be disseminated using easily accessible summary reports and resources to primary end users. PROSPERO REGISTRATION NUMBER: This protocol has been registered on PROSPERO (CRD42019130195).
Asunto(s)
Familia , Prevención del Suicidio , Revisiones Sistemáticas como Asunto , Humanos , Guías de Práctica Clínica como Asunto , Proyectos de InvestigaciónRESUMEN
INTRODUCTION: Pilot/feasibility studies assess the feasibility of conducting a larger study. Although researchers ought to communicate the feasibility objectives to their participants, many research ethics guidelines do not comment on how informed consent applies to pilot studies. It is unclear whether researchers and research ethics boards clearly communicate the purpose of pilot studies to participants consenting.The primary objective of this study is to assess whether pilot/feasibility studies submitted for ethics approval to a research ethics board transparently communicate the purpose of the study to participants through their informed consent practice. A highly transparent consent practice entails the consent documents communicate: (1) the term 'pilot' or 'feasibility' in the title; (2) the definition of a pilot/feasibility study; (3) the primary objectives of the study are to assess feasibility; (4) the specific feasibility objectives; and (5) the criteria for the study to successfully lead to the main study. The secondary objectives are to assess whether there is a difference between submitted and revised versions of the consent documents (revisions are made to obtain research ethics approval), to determine factors associated with transparent consent practices and to assess the consistency with which pilot and feasibility studies assess feasibility outcomes as their primary objectives. METHODS AND ANALYSIS: This is a retrospective review of informed consent information for pilot/feasibility studies submitted to the Hamilton integrated Research Ethics Board, Canada. We will look at submitted and revised consent documents for pilot/feasibility studies submitted over a 14-year period. We will use descriptive statistics to summarise data, reporting results as percentages with 95% CIs, and conduct logistic regression to determine characteristics associated with transparent consent practices. ETHICS AND DISSEMINATION: The study protocol was approved by the Hamilton integrated Research Ethics Board, and the results of this study will be submitted for publication in a peer-reviewed journal.
Asunto(s)
Consentimiento Informado/normas , Garantía de la Calidad de Atención de Salud , Estudios de Factibilidad , Humanos , Proyectos Piloto , Estudios RetrospectivosRESUMEN
Cutaneous involvement of multiple myeloma (MM) is uncommon, typically occurs in late stage disease, and is a poor prognostic indicator with an approximate eight month median survival. We present a 51-year-old man with relapsed lambda light chain MM who developed abrupt asymptomatic skin metastases. Biopsy revealed a dermis replete of atypical plasma cells, positive for CD138 and CD45. In situ hybridization confirmed lambda light chain restriction. Despite rescue antimyeloma therapy with the anti-CD38 drug daratumumab, he rapidly declined clinically and succumbed to the disease four weeks after presentation. A standard treatment approach for cutaneous MM does not currently exist; however, various techniques to detect cytogenetic abnormalities are emerging and will provide additional prognostic value and direct individualized therapy.
RESUMEN
Phosphoinositide 3-kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K-PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K-activated Akt signaling is well established in HNSCC, the significance of mTORC2-driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2-specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.
Asunto(s)
Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Antineoplásicos/farmacología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Cisplatino/farmacología , Clorhidrato de Erlotinib/farmacología , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismoRESUMEN
OBJECTIVES: The PI3K/AKT/mTOR pathway is frequently altered in head and neck squamous cell cancer (HNSCC), making this pathway a logical therapeutic target. However, PI3K targeting is not universally effective. Biomarkers of response are needed to stratify patients likely to derive benefit and exclude those unlikely to respond. MATERIALS AND METHODS: We examined the sensitivity of cell lines with constitutively-active (G12V mutant) HRAS and wild-type HRAS to PI3K inhibition using flow cytometry and cell viability assays. We then overexpressed and silenced HRAS and measured sensitivity to the PI3K inhibitor BYL719. Immunoblotting was used to determine activation of the PI3K pathway. MEK and mTOR inhibitors were then tested in HRAS mutant cells to determine their efficacy. RESULTS: HRAS mutant cell lines were non-responsive to PI3K inhibition. Overexpression of HRAS led to reduced susceptibility to PI3K inhibition, while knockdown improved sensitivity. Immunoblotting revealed suppressed AKT phosphorylation upon PI3K inhibition in both wild-type and HRAS mutant cell lines, however mutant lines maintained phosphorylation of S6, downstream of mTOR. Targeting mTOR effectively reduced viability of HRAS mutant cells and we subsequently examined the ERK-TSC2-mTOR cascade as a mediator of resistance to PI3K inhibition. CONCLUSIONS: HRAS mutant cells are resistant to PI3K inhibition and our findings suggest the involvement of a signalling intersection of the MAPK and PI3K pathways at the level of ERK-TSC2, leading to persistent mTOR activity. mTOR inhibition alone or in combination with MAPK pathway inhibition may be a promising therapeutic strategy for this subset of HNSCC tumors.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Tiazoles/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa/fisiología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Genes ras , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/fisiopatología , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Terapia Molecular Dirigida , Mutación Missense , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/fisiopatología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
BACKGROUND: The anti-tumor properties of cannabinoids have been investigated in many in vitro and in vivo studies. Many of these anti-tumor effects are mediated via cannabinoid receptor types 1 and 2 (CB1 and CB2), comprising the endocannabinoid system (ECS). In this study, we investigated the ECS based on CB 1 and CB 2 receptor gene and protein expression in renal cell carcinoma (RCC) cell lines. In view of their further use for potential treatments, we thus investigated the roles of CB1 and CB2 receptors in the anti-proliferative action and signal transduction triggered by synthetic cannabinoid agonists [such as JWH-133 and WIN 55,212-2 (WIN-55)] in RCC cell lines. METHODS: Human RCC cell lines were used for this study. The CB 1 and CB 2 gene expression levels were analyzed using real-time PCR. Flow cytometric, immunocytochemical and western blot analyses were performed to confirm CB1 and CB2 receptor protein expression. The anti-proliferative effects of synthetic cannabinoids were investigated on cell viability assay. The CB1 and CB2 receptors were blocked pharmacologically with the antagonists SR141716A and AM-630, respectively, to investigate the effects of the agonists JWH-133 and WIN-55. Cell cycle, apoptosis and LDH-based cytotoxicity were analyzed on cannabinoid-treated RCC cells. RESULTS: The CB1 and CB2 genes expression was shown by real-time PCR and flow cytometric and western blot analysis indicating a higher level of CB2 receptor as compared to CB1 in RCC cells. Immunocytochemical staining also confirmed the expression of the CB1 and CB2 proteins. We also found that the synthetic cannabinoid agonist WIN-55 exerted anti-proliferative and cytotoxic effects by inhibiting the growth of RCC cell lines, while the CB2 agonist JWH-133 did not. Pharmacologically blocking the CB1 and CB2 receptors with their respective antagonists SR141716A and AM-630, followed by the WIN-55 treatment of RCC cells allowed uncovering the involvement of CB2, which led to an arrest in the G0/G1 phase of the cell cycle and apoptosis. CONCLUSIONS: This study elucidated the involvement of CB2 in the in vitro inhibition of RCC cells, and future applications of CB2 agonists in the prevention and management of RCC are discussed.
Asunto(s)
Benzoxazinas/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Morfolinas/farmacología , Naftalenos/farmacología , Receptor Cannabinoide CB2/metabolismo , Apoptosis/efectos de los fármacos , Benzoxazinas/uso terapéutico , Agonistas de Receptores de Cannabinoides/uso terapéutico , Antagonistas de Receptores de Cannabinoides/farmacología , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Renales/patología , Morfolinas/uso terapéutico , Naftalenos/uso terapéutico , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
The aim of this study was to compare effect of everolimus on growth of different renal cell carcinoma (RCC) populations and develop experimental design to measure the early response of everolimus in clear cell RCC (ccRCC) cell lines including renal cancer stem cells. Effect of everolimus on RCC cell lines which include primary (786-0) and metastatic (ACHN) RCC cell lines as well as heterogenous populations of tumor cells of different histological RCC subtypes (clear cell RCC and papillary RCC) was measured when treated with everolimus in the range of 1-9 µM. Gene expression profiling using microarray was performed to determine the early response to everolimus in ccRCC cell lines after optimizing concentration of drug. Gene Set Enrichment Analysis (GSEA) was done which mainly focused on basic genes related to mTOR, hormonal and metabolic pathways. Everolimus acts on RCC cells in a dose-dependent manner. In all examined cell lines IC50 dose was possible to calculate after the third day of treatment. In ccRCC lines (parental and stem cell) everolimus changes expression of mTOR complexes elements and elements of related pathways when treated with optimized doses of drug. Characteristic expression profile for ccRCC cells at an early exposure time to everolimus is to elucidate. Wevarie include some basic observations derived from data analysis in the context of mechanism of action of drug with a view to better understand biology of renal cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Renales/metabolismo , Everolimus/farmacología , Neoplasias Renales/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
BACKGROUND: Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. MATERIALS AND METHODS: Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. RESULTS: Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-ß, Wnt/ß-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells. CONCLUSIONS: All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Endoglina/metabolismo , Perfilación de la Expresión Génica , Neoplasias Renales/genética , Neoplasias Renales/patología , Células Madre Neoplásicas/citología , Antígeno AC133/metabolismo , Biomarcadores de Tumor/metabolismo , Transición Epitelial-Mesenquimal , Humanos , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
In recent years, genome-wide RNA expression analysis has become a routine tool that offers a great opportunity to study and understand the key role of genes that contribute to carcinogenesis. Various microarray platforms and statistical approaches can be used to identify genes that might serve as prognostic biomarkers and be developed as antitumor therapies in the future. Metastatic renal cell carcinoma (mRCC) is a serious, life-threatening disease, and there are few treatment options for patients. In this study, we performed one-color microarray gene expression (4×44K) analysis of the mRCC cell line Caki-1 and the healthy kidney cell line ASE-5063. A total of 1,921 genes were differentially expressed in the Caki-1 cell line (1,023 upregulated and 898 downregulated). Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) approaches were used to analyze the differential-expression data. The objective of this research was to identify complex biological changes that occur during metastatic development using Caki-1 as a model mRCC cell line. Our data suggest that there are multiple deregulated pathways associated with metastatic clear cell renal cell carcinoma (mccRCC), including integrin-linked kinase (ILK) signaling, leukocyte extravasation signaling, IGF-I signaling, CXCR4 signaling, and phosphoinositol 3-kinase/AKT/mammalian target of rapamycin signaling. The IPA upstream analysis predicted top transcriptional regulators that are either activated or inhibited, such as estrogen receptors, TP53, KDM5B, SPDEF, and CDKN1A. The GSEA approach was used to further confirm enriched pathway data following IPA.