Migration, invasion, and metastasis are mediated by P-Rex1 in neuroblastoma.

Neuroblastoma accounts for approximately 15% of pediatric cancer-related deaths despite intensive multimodal therapy. This is due, in part, to high rates of metastatic disease at diagnosis and disease relapse. A better understanding of tumor biology of aggressive, pro-metastatic phenotypes is necessary to develop novel, more effective therapeutics against neuroblastoma. Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) has been found to stimulate migration, invasion, and metastasis in several adult malignancies. However, its role in neuroblastoma is currently unknown. In the present study, we found that P-Rex1 is upregulated in pro-metastatic murine models of neuroblastoma, as well as human neuroblastoma metastases. Correspondingly, silencing of P-Rex1 was associated with decreased migration and invasion in vitro. This was associated with decreased AKT-mTOR and ERK2 activity, dysregulation of Rac, and diminished secretion of matrix metalloproteinases. Furthermore, increased P-Rex1 expression was associated with inferior relapse-free and overall survival via tissue microarray and Kaplan-Meier survival analysis of a publicly available clinical database. Together, these findings suggest that P-Rex1 may be a novel therapeutic target and potential prognostic factor in neuroblastoma.

Social Determinants of Outcomes Disparity among Pediatric Patients with Solid Tumor.

BACKGROUND: Socioeconomic factors have a significant impact on healthcare outcomes. Metrics such as area deprivation index (ADI) are used to quantify the anticipated influence of these factors. Here, we sought to assess the impact of socioeconomic factors on clinical outcomes among pediatric patients with solid tumor in our region. STUDY DESIGN: We identified 3,863 pediatric patients who were diagnosed with a malignant solid tumor in the Texas Cancer Registry between 1995 and 2019. ADI was used to quantify socioeconomic determinants of health. These outcome variables were determined: stage of disease at diagnosis, time between diagnosis and treatment initiation, and overall mortality. Statistical analysis was performed using logistic regression, linear regression, Cox proportional hazards regression, and Kaplan-Meier survival curves. RESULTS: A total of 53.5% of patients were male and the average age at diagnosis was 4.5 years. Forty-seven percent of patients were White, 13.3% were Black, 36.2% were Hispanic, 1.7% were Asian, and other rare minority groups made up 1.8%. On multivariable analysis, increased risk of death was associated with Black race, rare minority race, residence in a border county, and increasing ADI score, with the risk of death at 5 years rising 4% with each increasing ADI point. CONCLUSIONS: Social determinants of health are associated with disparate outcomes among pediatric patients with solid tumor. Our results suggest that patients who are part of racial minority groups and those who reside in socioeconomically disadvantaged neighborhoods or regions near the Texas-Mexico border are at an increased risk of death. This information may be useful in strategizing outreach and expanding resources to improve outcomes in at-risk communities.

WDR5 facilitates recruitment of N-MYC to conserved WDR5 gene targets in neuroblastoma cell lines.

Collectively, the MYC family of oncoprotein transcription factors is overexpressed in more than half of all malignancies. The ability of MYC proteins to access chromatin is fundamental to their role in promoting oncogenic gene expression programs in cancer and this function depends on MYC-cofactor interactions. One such cofactor is the chromatin regulator WDR5, which in models of Burkitt lymphoma facilitates recruitment of the c-MYC protein to chromatin at genes associated with protein synthesis, allowing for tumor progression and maintenance. However, beyond Burkitt lymphoma, it is unknown whether these observations extend to other cancers or MYC family members, and whether WDR5 can be deemed as a "universal" MYC recruiter. Here, we focus on N-MYC amplified neuroblastoma to determine the extent of colocalization between N-MYC and WDR5 on chromatin while also demonstrating that like c-MYC, WDR5 can facilitate the recruitment of N-MYC to conserved WDR5-bound genes. We conclude based on this analysis that N-MYC and WDR5 colocalize invariantly across cell lines at predicted sites of facilitated recruitment associated with protein synthesis genes. Surprisingly, we also identify N-MYC-WDR5 cobound genes that are associated with DNA repair and cell cycle processes. Dissection of chromatin binding characteristics for N-MYC and WDR5 at all cobound genes reveals that sites of facilitated recruitment are inherently different than most N-MYC-WDR5 cobound sites. Our data reveals that WDR5 acts as a universal MYC recruiter at a small cohort of previously identified genes and highlights novel biological functions that may be coregulated by N-MYC and WDR5 to sustain the neuroblastoma state.

High-Risk Neuroblastoma: A Surgical Perspective.

High-risk neuroblastoma requires multimodal treatment including systemic chemotherapy, surgical resection, radiation therapy, stem cell transplant, and immunotherapy. Surgeons play a vital role in obtaining local control of neuroblastoma and must therefore be knowledgeable about this complex pathology. This article provides a review of the optimal timing and extent of resection, the impact of various image-defined risk factors on surgical planning, and surgical approaches and techniques to enhance the resection of tumors in different anatomic locations.

Improving Billing and Collections in a High-Volume Pediatric Surgery Practice: Denials-Based Approach.

BACKGROUND: Despite minimal coding and billing training, surgeons are frequently tasked with both in clinical practice. This often results in denials for reimbursement based on incorrect or insufficient documentation, and reduced collections for work performed. We sought to evaluate how to correct these deficits while improving reimbursement for the most frequently rejected procedures at a high-volume academic center. STUDY DESIGN: Hospital billing data were analyzed for a 4-year period (2018 to 2021) to determine the CPT code denials with the largest overall cost. The denials were then stratified according to payor, reason for denial, and preventability. Assigned ICD-10 codes were categorized based on specificity as related to the procedure. The distribution of denials according to ICD-10 specificity was evaluated using the chi-square test. RESULTS: A total of 8,833 denials representing $11,009,108 in billing were noted during the study period. The CPT code 44970 (laparoscopic appendectomy) was the code associated with the largest financial impact, representing 12.8% of the total denied amount ($1.41M). Of the 823 denials for CPT 44970, 93.3% were associated with nonspecific ICD-10 codes, whereas only 42.0% had been associated with procedure-specific ICD-10 codes. Of the patients with nonspecific codes, 80.7% of denials were due to criteria that could be remedied with supplemental information or timely filing, representing $1,059,968 in collections. CONCLUSIONS: This is the first study to systematically evaluate a pathway for using denial data to improve collections for work performed at a high-volume academic pediatric surgery practice. Using this methodology, targets for improvement in coding and/or documentation can be identified to improve the financial performance of a surgical department. This study also provides evidence that association with nonspecific diagnostic codes is correlated with initial denial of payment by insurance companies.

Anti-GRP-R monoclonal antibody antitumor therapy against neuroblastoma.

Standard treatment for patients with high-risk neuroblastoma remains multimodal therapy including chemoradiation, surgical resection, and autologous stem cell rescue. Immunotherapy has demonstrated success in treating many types of cancers; however, its use in pediatric solid tumors has been limited by low tumor mutation burdens. Gastrin-releasing peptide receptor (GRP-R) is overexpressed in numerous malignancies, including poorly-differentiated neuroblastoma. Monoclonal antibodies (mAbs) to GRP-R have yet to be developed but could serve as a potential novel immunotherapy. This preclinical study aims to evaluate the efficacy of a novel GRP-R mAb immunotherapy against neuroblastoma. We established four candidate anti-GRP-R mAbs by screening a single-chain variable fragment (scFv) library. GRP-R mAb-1 demonstrated the highest efficacy with the lowest EC50 at 4.607 ng/ml against GRP-R expressing neuroblastoma cells, blocked the GRP-ligand activation of GRP-R and its downstream PI3K/AKT signaling. This resulted in functional inhibition of cell proliferation and anchorage-independent growth, indicating that mAb-1 has an antagonist inhibitory role on GRP-R. To examine the antibody-dependent cellular cytotoxicity (ADCC) of GRP-R mAb-1 on neuroblastoma, we co-cultured neuroblastoma cells with natural killer (NK) cells versus GRP-R mAb-1 treatment alone. GRP-R mAb-1 mediated ADCC effects on neuroblastoma cells and induced release of IFNγ by NK cells under co-culture conditions in vitro. The cytotoxic effects of mAb-1 were confirmed with the secretion of cytotoxic granzyme B from NK cells and the reduction of mitotic tumor cells in vivo using a murine tumor xenograft model. In summary, GRP-R mAb-1 demonstrated efficacious anti-tumor effects on neuroblastoma cells in preclinical models. Importantly, GRP-R mAb-1 may be an efficacious, novel immunotherapy in the treatment of high-risk neuroblastoma patients.

Combination bromo- and extraterminal domain and poly (ADP-ribose) polymerase inhibition synergistically enhances DNA damage and inhibits neuroblastoma tumorigenesis.

PURPOSE: JQ1 is a bromo- and extraterminal (BET) domain inhibitor that downregulates MYC expression and impairs the DNA damage response. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent DNA damage sensing and repair. We hypothesized that JQ1 would promote a DNA repair-deficient phenotype that sensitizes neuroblastoma cells to PARP inhibition. METHODS: Four human neuroblastoma cell lines were examined: two MYCN-amplified (BE(2)-C and IMR-32), and two non-MYCN-amplified (SK-N-SH and SH-SY5Y). Cells were treated with JQ1 (BET inhibitor), Olaparib (PARP inhibitor), or in combination to assess for therapeutic synergy of JQ1 and Olaparib. Treated cells were harvested and analyzed. Quantitative assessment of combination treatment synergy was performed using the median effect principle of Chou and Talalay. RESULTS: Combination treatment with Olaparib decreased the IC50 of JQ1 by 19.9-fold, 2.0-fold, 12.1-fold, and 2.0-fold in the BE(2)-C, IMR-32, SK-N-SH, and SH-SY5Y cell lines, respectively. In the MYCN-amplified cell lines, BE(2)-C and IMR-32, combination treatment decreased gene expression of MYCN relative to single-drug treatment alone or control. Combination treatment decreased protein expression of DNA repair proteins Ku80 and RAD51, led to accumulation of DNA damage marker phospho-histone H2A.X, and increased caspase activity. In the non-MYCN-amplified cell lines, SK-N-SH and SH-SY5Y, combination treatment induced G0/G1 cell cycle arrest. CONCLUSIONS: Combination BET and PARP inhibition synergistically inhibited neuroblastoma tumorigenesis in vitro. In MYCN-amplified neuroblastoma cells, this effect may be induced by downregulation of MYCN transcription, defects in DNA repair, accumulation of DNA damage, and apoptosis. In non-MYCN-amplified cell lines, combination treatment induced cell cycle arrest.

Ethnic Disparities in the Management of Pediatric Subcutaneous Abscesses.

(1) Background: Significant racial and ethnic disparities affect access to pediatric Emergency Department (ED) and surgical care across the United States. The present study sought to assess the role of racial and ethnic disparities in the management of pediatric subcutaneous abscesses. (2) Methods: A retrospective chart review was performed including ED visits for subcutaneous abscesses in patients < 18 years of age, over a 12-month period. The effects of self-reported ethnicity (Hispanic versus non-Hispanic) and race (Hispanic, Black, Caucasian and Asian) on the diagnosis and management of subcutaneous abscesses were analyzed. (3) Results: 192 patients were identified with an average age of 4.7 ± 5.3 years and 43.8% identified as Hispanic. Non-Hispanic patients were significantly more likely to receive treatment of their SSTI prior to the ED and to be admitted, compared to Hispanic patients. There was no difference in bedside versus operating room incision and drainage (I&D); however, significantly more non-Hispanic patients received procedural sedation for bedside I&D compared to Hispanic patients. There were no differences in outcomes such as recurrence or re-admission based on ethnicity or race. (4) Conclusions: Ethnic and racial disparities exist in the management of subcutaneous abscesses in the United States. Further studies are needed to address the systemic causes of these disparities such as access to tertiary healthcare facilities and systems-based analyses of unconscious bias in healthcare.

Multimodality treatment of pediatric Ewing sarcoma: A single-center 10-year analysis of outcomes.

BACKGROUND: Ewing sarcoma, a malignancy originating from the bone or soft tissues most commonly diagnosed in adolescents, requires multimodality therapy. Although both surgical resection and radiation therapy are effective local control modalities, there are limited data comparing outcomes in patients treated with surgery versus radiation. We sought to determine whether there were differences in 5-year local failure-free survival, event-free survival, and overall survival based on the modality used for local control. METHODS: Patients treated for Ewing sarcoma at a single tertiary pediatric hospital between 2010 and 2020 were included for retrospective analysis. Patient and tumor demographics, treatment information, and patient response to therapies were collected from the medical record. Outcome measures were local failure-free survival, event-free survival, and overall survival at 5 years from diagnosis. RESULTS: Sixty-one patients met inclusion criteria. All patients received chemotherapy, and 68.9% of patients presented with localized disease. Of these, 23.8% were treated with radiation alone; the remaining 76.2% underwent resection ± radiation. A total of 52.4% of patients with localized disease achieved R0 resection. Only 3 patients experienced local progression; there was no difference between treatment groups. There was no significant association between local control modality and event-free survival or overall survival in patients with localized disease, regardless of margin status. CONCLUSION: There was no significant difference in 5-year local failure-free survival, event-free survival, or overall survival in Ewing sarcoma patients treated with radiation versus surgery ± radiation, regardless of whether or not R0 resection was achieved. Future directions include a multi-institutional study to allow for further subgroup analysis and increased sample size.

Impact of Cryoablation on Pectus Excavatum Repair in Pediatric Patients.

BACKGROUND: Minimally invasive repair of pectus excavatum (MIRPE) involves placement of a transthoracic, retrosternal support bar under thoracoscopic guidance. Despite its minimally invasive technical approach, postoperative pain is a significant morbidity that often results in increased length of stay. Multi-modal pain control strategies have been used in the past with limited success. Recently, the use of intraoperative intercostal nerve cryoablation (CA) has been added. In the present study, we aim to evaluate the effects of CA on postoperative pain control, opioid requirements, and perioperative outcomes. STUDY DESIGN: A single-center, retrospective chart review of all patients (less than 18 years old) who underwent MIRPE from 2009 to 2020 was performed. CA was started in June 2018. Data collection included demographics, preoperative characteristics, intraoperative findings, and postoperative outcomes. We hypothesized that CA would be associated with improved pain scores, lower doses of total inpatient opioid requirement, and shorter length of stay (LOS). RESULTS: One hundred sixty-one patients met inclusion criteria: 75 underwent intraoperative CA and 86 underwent MIRPE without CA (NCA group). CA significantly decreased median LOS from 4 days in NCA to 2 days; the use of CA was the only significant predictor of LOS on linear regression. CA was also associated with decreased total PCA, intravenous opioid, and oral opioid dosages. There was no difference in inpatient pain scores and a slight increase in mean procedure time. However, CA was associated with significantly decreased postoperative complications. CONCLUSIONS: The use of cryoablation during MIRPE significantly decreases LOS, perioperative opioid requirements, and postoperative complications, with a minimal increase in operative time. Cryoablation is an effective pain control modality in the surgical management of chest wall deformities in children.

Effect of Quarantine and Reopening Measures on Pediatric Trauma Admissions During the 2019 SARS-CoV2 Virus Pandemic.

BACKGROUND: Several studies have reported decreased trauma admissions and increased physical abuse in children resulting from stay-at-home measures. However, these studies have focused on a limited period after the implementation of lockdown policies. The purpose of this study was to examine the effect of quarantine and reopening initiatives on admissions for varying types of injuries in pediatric patients. STUDY DESIGN: Registry data for an urban Level I pediatric trauma center were evaluated from April 1, 2018, to March 30, 2021. A timeline of local shutdown and reopening measures was established and used to partition the data into 6-month intervals. Data about demographics and injury characteristics were compared with similar intervals in 2018 and 2019 using appropriate statistical methodology for categorical, parametric, and nonparametric data. RESULTS: A total of 3,110 patients met criteria for inclusion. A total of 1,106 patients were admitted the year after the closure of schools and nonessential businesses. Decreases in overall admissions and evaluations for suspected child abuse noted early in the pandemic were not sustained during shutdown or reopening periods. However, we observed a 77% increase in all-terrain vehicle injuries, along with a 59% reduction in sports injuries (chi-square [8, N = 3,110] = 49.7; p < 0.001). Significant shifts in demographic and payor status were also noted. CONCLUSIONS: This is the first study to comprehensively examine the effects of quarantine and reopening policies on admission patterns for a pediatric trauma center in a metropolitan area. Total admissions and child abuse evaluations were not impacted. If shutdown measures are re-instituted, preventative efforts should be directed towards ATV use and recreational activities.

Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma.

High-risk neuroblastoma (NB) remains an extremely difficult subgroup to cure and is associated with MYCN amplification. Serine hydroxymethyltransferase 2 (SHMT2) regulates serine metabolism in a myc-dependent manner; it is upregulated in several cancers and is associated with tumor aggressiveness. Akt-2, an important regulator of MYCN via the PI3K/Akt pathway, induces metastatic potential in NB. The association between SHMT2 and PI3K/Akt in hepatocyte regeneration has been well established but its mechanistic interaction in cancer has yet to be clearly elucidated. Herein, we evaluated the exact role of SHMT2 on the PI3K/Akt pathway, in addition to NB tumorigenesis and metastatic potential in vitro. SHMT2 gene expression and overall survival (OS) were assessed. Two human NB cell lines were examined. SHMT2 silencing and overexpression were performed. The downstream effects were analyzed with immunoblotting, RT-qPCR and functional assays were performed. We found SHMT2 gene expression is associated with decreased OS and MYCN amplification. SHMT2 protein and mRNA expression are increased in MYCN-amplified cells. SHMT2 expression has a direct interaction with Akt-2 and MYCN. Induction of SHMT2 increased cellular proliferation, colony formation and cellular migration and SHMT2 expression was increased in metastatic NB cells. We conclude that SHMT2 regulates N-Myc via phosphorylation of Akt-2 and plays an important role in NB tumorigenesis by contributing to cell growth, migration, colony formation and metastasis in vitro.

Reactivation of silenced α-N-catenin induces retinoic acid sensitivity in neuroblastoma cells.

BACKGROUND: High-risk neuroblastoma remains the most difficult pediatric solid tumor to treat and is associated with chemotherapy and radiation resistance that may be secondary to epigenetic modifications. We have previously found that α-N-catenin, a cell-adhesion protein encoded by the gene CTNNA2, plays a tumor suppressor role in neuroblastoma by inhibiting the NF-κB signaling pathway. A subset of neuroblastoma tumors that lack α-N-catenin are resistant to all-trans retinoic acid. However, the mechanism of CTNNA2 silencing in neuroblastoma remains unknown. Herein, we sought to determine the mechanism of α-N-catenin silencing in neuroblastoma. METHODS: Two human neuroblastoma cell lines, SK-N-AS and BE(2)-C, were stably transfected with a plasmid expressing CTNNA2. Both cell lines were treated with the histone deacetylase inhibitor Trichostatin A alone and in combination with retinoic acid. Cell survival and colony formation were measured. Cellular differentiation and expression of cell survival signaling pathways were analyzed. Immunoblotting and reverse transcription quantitative polymerase chain reaction were used to examine protein and messenger RNA expression. RESULTS: Retinoic acid treatment induced cellular differentiation and inhibited cellular proliferation in BE(2)-C cells but did not induce differentiation in SK-N-AS cells. Re-expression of α-N-catenin enhanced the sensitivity to retinoic acid-induced cell growth arrest and downregulated key cell survival pathways in both cell lines. Trichostatin A treatment induced CTNNA2 expression in SK-N-AS cells, and combination treatment with Trichostatin A induced retinoic acid sensitivity in retinoic acid-resistant cells. CONCLUSION: Re-expression of α-N-catenin in retinoic acid-resistant cells induced sensitivity to retinoic acid treatment and is controlled epigenetically via histone deacetylase. α-N-catenin is a potential biomarker for retinoic acid sensitivity and combination treatment with Trichostatin A and retinoic acid may improve survival among children with high-risk, retinoic acid-resistant neuroblastoma.

Pediatric Subcutaneous Abscess: Still a Clinical Exam-Based Diagnosis and Treatment.

Subcutaneous abscesses occur frequently in the pediatric population, yet there is great variability in the approach to diagnosis and management, partly due to opposing recommendations in the current literature and the lack of a standardized protocol for diagnosis and management among pediatric medical centers. This has led to inconsistencies by the providers, as well as the hospital clinical pathways, with regards to the appropriate management of subcutaneous abscesses. We hypothesize that the current variability in diagnostic work-up and management contributes to the wide use of unnecessary imaging and therapeutics without altering the overall outcomes. We performed a retrospective chart review that compared 200 encounters for patients < 18 years of age with a diagnosis of subcutaneous abscess at a single large tertiary pediatric institution. Our results showed that only 13.6% of wound cultures obtained led to a change in the antibiotic regimen and that blood cultures were positive in only 2.1% of cases. There was no difference in the incision and drainage performed based on ultrasound findings in the presence of fluctuance on physical exam. Patients presenting with fever were more likely to be admitted to the hospital for further care than those without fever. Our results showed no difference in outcome after incision and drainage for abscesses packed with gauze versus those left to drain via a vessel loop drain. There was no difference in recurrence in patients discharged with oral antibiotics versus without oral antibiotic treatment. Our data indicate that many of the diagnostic studies used for the management of a subcutaneous abscess have little to no effect on the outcome. Subcutaneous abscesses are a common pediatric complaint, and this study could help healthcare providers utilize more effective and efficient management strategies for skin and soft tissue infections.

Predictive Factors and Outcomes for Successful Thoracoscopic Lung Resection in Pediatric Patients.

BACKGROUND: Less than 50% of children with congenital lung lesions are treated thoracoscopically. There are variable data regarding the benefits and limited information on factors contributing to successful thoracoscopic lobectomies in pediatric patients. We sought to identify predictive factors leading to safe and efficient thoracoscopic lung resection. STUDY DESIGN: We performed a single-center, retrospective chart review of patients (age <18 y) who underwent lung resection between June 2009 and July 2020. Pulmonary wedge resection was excluded. Data collected included demographics, perioperative findings, such as symptoms or infection, and postoperative outcomes. Univariate, multivariate, and sensitivity analyses were performed. RESULTS: Ninety-six patients were identified. Sixty-nine patients (72%) underwent initial thoracoscopy, with 15 (22%) converting to open thoracotomy (CTO). Forty-one (43%) patients had preoperative symptoms and 15 (15.6%) had an active infection. Among symptomatic patients, 18 (43.9%) underwent thoracotomy and 23 (56%) were attempted thoracoscopically, 13 (31%) of whom were completed thoracoscopically. On univariate analysis, age >1 year, infection, preoperative symptoms, and intraoperative adhesions were associated with CTO. Older age (odds ratio [OR] = 1.041) and estimated blood loss (EBL) (OR = 2.398) were significant prognostic factors of CTO on logistic regression. Thoracoscopy was significantly associated with decreased length of stay, opioid use, chest tube duration, blood loss and need for blood transfusion. There was no difference in operative time, 30-day readmission, or mortality. CONCLUSIONS: Thoracoscopy has become a standard approach for pediatric lung resection. Our findings indicate that age < 1 year and the absence of active respiratory infection and preoperative symptoms may be predictive of successful completion of the thoracoscopic approach. Thoracoscopy offers significant advantages over the traditional open thoracotomy with regard to blood loss and opioid requirements, LOS, and chest tube duration.

Preclinical evaluation of the anti-tumor activity of pralatrexate in high-risk neuroblastoma cells.

INTRODUCTION: Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier-1 with antineoplastic and immunosuppressive activities, similar to methotrexate. Despite advances in multi-modality treatment strategies, the survival rates for children with high-risk neuroblastoma have failed to improve. Therefore, the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat high-risk neuroblastoma. MATERIALS AND METHODS: Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents. Antiproliferative effects of pralatrexate were assessed by adherent and non-adherent colony formation assays. Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting. PDX tissue culture was used to assess ex vivo efficacy. RESULTS: Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a time-dependent manner. The potency of pralatrexate was ten-fold stronger than methotrexate, as measured by IC50. Pralatrexate-induced apoptosis was confirmed by caspase-3 activation and PARP cleavage. MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCN-amplified neuroblastoma cells. CONCLUSIONS: Pralatrexate demonstrated effective inhibition of cell growth and viability. The higher potency of pralatrexate compared to methotrexate, a drug with high levels of toxicity, suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with high-risk neuroblastoma.

Overexpression of microRNA-145 inhibits tumorigenesis through autophagy in chemotherapy and radiation resistant neuroblastoma cells.

MicroRNA-145 (miR-145) plays a suppressive role in the process of tumorigenesis and an important role in induction of autophagy. However, the exact role of miR-145 in therapeutically resistant neuroblastoma cells remain elusive. Herein, we sought to evaluate the effects of miR-145 overexpression in chemo­ and radiation-resistant neuroblastoma cells. We hypothesized that miR-145 affects the aggressiveness of resistant cells by enhancing autophagy. We established Cisplatin-resistant (CDDP-R), Vincristine-resistant (Vin-R), and radiation-resistant (Rad-R) neuroblastoma cells and found that miR-145 expression was significantly decreased in the resistant cells compared to the parental cells. Exogenously expression of miR-145 inhibited oncogenic properties such as proliferation, clonogenicity, anchorage-independent growth, cell migration, and tubule formation in the resistant cells. In addition, we also found that an autophagy protein marker, LC3, was only minimally expressed in the resistant cells. In particular, when miR-145 was overexpressed in the resistant cells, LC3 I and II were expressed and an increased punctate fluorescence of LC3 protein was found indicating the induction of autophagy. Taken together, our data suggests that miR-145 inhibits tumorigenesis and aggressiveness via modulation of autophagy in neuroblastoma.